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

  1. Miae Jang
  2. Elizabeth Gould
  3. Jie Xu
  4. Eun Jung Kim
  5. Jun Hee Kim  Is a corresponding author
  1. University of Texas Health Science Center, United States
  2. The Second Xiangya Hospital, Central South University, China
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Cite this article as: eLife 2019;8:e42156 doi: 10.7554/eLife.42156

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 glutamate vesicles, without altering presynaptic Ca2+ 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.

https://doi.org/10.7554/eLife.42156.001

Introduction

The formation of complex neuronal networks requires the experience-dependent establishment and remodeling of synapses. The precise control of synaptic function depends not only on neurons, but also on glial cells. Immature oligodendrocytes, located near synapses, make functional synapses with 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, 2009). Thus, oligodendrocytes are in a prime position to participate in bi-directional communication. In this study, we address whether oligodendrocytes can modulate synaptic function through activity-dependent BDNF signaling.

BDNF regulates neuronal survival and growth in the developing central nervous system (CNS; Alderson et al., 1990; Hohn et al., 1990; Rodriguez-Tébar et al., 1990) and is extensively involved 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 involved in long-term synaptic plasticity in the hippocampus (Harward et al., 2016; Vignoli et al., 2016; Gärtner and Staiger, 2002). It is known that BDNF signaling regulates vesicular glutamate release at presynaptic terminals (Pozzo-Miller et al., 1999; Tyler and Pozzo-Miller, 2001), but, because of the low expression of both intracellular and extracellular BDNF in most brain areas, little is known regarding the source and the exact location of action of BDNF at synapses and, specifically, its presynaptic effects.

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 Dreyfus, 2009). We recently showed that immature oligodendrocytes have the capacity to sense neuronal 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 conditional 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 brainstem response (ABR) tests, we found that oligodendroglial BDNF is critical for determining the readily 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 brainstem 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 glutamatergic transmission in the auditory brainstem, we recorded miniature excitatory post-synaptic currents (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 (τ)=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 endogenous BDNF results in impaired glutamatergic transmission, which is caused by alterations in presynaptic properties rather than postsynaptic components.

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 μm. (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 (τ, 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).

https://doi.org/10.7554/eLife.42156.002

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 does not alter the Ca2+-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 displayed 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 (Pr), 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 second through the fourth EPSC) and extrapolating to the x axis, we measured the total equivalent 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 difference 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).

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 (Pr) using the EQ method in WT and Bdnf+/– mice. (F) Plot of the cumulative 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 (Pr) 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).

https://doi.org/10.7554/eLife.42156.003

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-intercept 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 (Pr), 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, suggesting 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 stimulation (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 neurotransmitter and Ca2+ influx at presynaptic terminals by measuring the membrane capacitance jump (ΔCm) and voltage-activated Ca2+ channel current (ICa) in P9–13 calyces from WT and Bdnf+/− mice (Figure 3A). Depolarization induced Ca2+ currents and, consequently, ΔCm in a pulse duration–dependent manner (2 to 20 ms). ΔCm in Bdnf+/− mice was much smaller than that in WT (ΔCm after a 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, unpaired t-test; Figure 3C), indicating that the sizes of the calyx terminals are similar, and thus the difference in ΔCm was not due to the terminal size. In addition, plotting ΔCm (for the 20 ms depolarization) as a function of the Cm showed the distribution of Cm as estimated by linear regression analysis (R2 = 0.1 in WT, n = 9; R2 = 0.06 in Bdnf+/– mice, n = 9; Figure 3C). Despite their similar resting values for Cm, ΔCm was much smaller in calyces from Bdnf+/− mice as compared with comparably sized calyces from WT. However, a positive correlation between the resting Cm and ΔCm suggest that larger calyces release more vesicles in both genotypes (Figure 3C). Next, we examined the effects of BDNF reduction on presynaptic Ca2+ channel and ICa related to changes in exocytosis of glutamate vesicles. There was no significant difference in the presynaptic ICa charge (QICa) in response to depolarizing pulses. The smaller ΔCm in calyces from Bdnf+/− mice was not attributed to alterations in presynaptic Ca2+ currents. In WT calyces, a 20 ms depolarization induced a ICa of 6.5 ± 0.53 pC/pF (n = 10), whereas in Bdnf+/− calyces, ICa was 5.9 ± 0.96 pC/pF (n = 9; p=0.50, unpaired t-test; Figure 3D). In addition, the current–voltage relationship (I–V) curve for these voltage-activated Ca2+ 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 amplitude and half-with of presynaptic action potential, which is associated with Ca2+ channel activation 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 μs in WT, n = 3 vs 283 ± 45.5 μs 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 in Ca2+ influx via Ca2+ channels at the presynaptic terminals.

Figure 3 with 1 supplement see all
Exocytosis of vesicular glutamate is decreased at the calyx terminals in Bdnf+/– mice.

(A) Representative traces of membrane capacitance (Cm; top) and Ca2+ current (ICa; middle) induced by 2-, 3-, 5-, 10-, and 20 ms depolarization (bottom) from P9–13 calyx terminals in WT (black) and Bdnf+/– (gray) mice. Capacitance within 50 ms after depolarization is not shown to avoid artifacts. (B) Summary of capacitance changes (ΔCm), which are plotted against the depolarization duration in WT and Bdnf+/– mice. (C) Scatter plot: ΔCm (elicited by 20 ms depolarization)is plotted against the corresponding resting Cm 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, respectively. (D) Summary of Ca2+ current charge (QICa), which is plotted against the depolarization duration for each genotype. (E) Representative traces of ICa induced by a 200 ms step-like depolarization (from –80 to 60 mV, Δ10 mV) in WT (black; n = 5) and Bdnf+/– (gray; n = 5) mice. (F) The I–V relationship for voltage-activated Ca2+ channels at the calyx terminal for each genotype is also shown. (G) Examples of Cm (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 (ΣΔCm) 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 ΣΔCm 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).

https://doi.org/10.7554/eLife.42156.004

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 (ΣΔCm), 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; Figure 3—figure supplement 1). Therefore, the decreased ΣΔCm 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; Marongiu et al., 2013), to determine whether this activation rescues the impaired RRP size and glutamate release at the calyx synapses in Bdnf+/– mice. In WT mice, the pre-application of 20 μM 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 control, 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 glutamatergic transmission in the MNTB.

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 μm. (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, 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).

https://doi.org/10.7554/eLife.42156.006

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 investigated 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 development (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 specifically deleted in CNPase-expressing oligodendrocytes using the Cre/loxP system (Figure 5B). To confirm the specificity of the Cnpcre line, Cnpcre 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—figure supplement 1). This confirms that Cnpcre is specific to oligodendrocytes and is not expressed in neurons or astrocytes in the MNTB of the auditory brainstem. In addition, CNP+ cells were positive for CC1, but negative for PDGFRa, indicating that most CNP+ cells in the MNTB are pre-myelinating oligodendrocytes beyond the precursor stage (Figure 5—figure supplement 1). Bdnf cKO mice (Cnpcre:Bdnffl/fl; Figure 5B) were generated by crossing Cnpcre mice with mice containing a floxed allele of BDNF (Bdnffl/fl). To further confirm the oligodendrocyte-specific depletion of BDNF, oligodendrocytes were isolated via the fluorescent activated cell sorting (FACS) using an O1 antibody, which is specific to oligodendrocytes, or Cnp- driven GCaMP6f-GFP (GFP). Utilizing quantitative PCR, we confirmed that the sorted O1+ or GFP+ fraction expressed a substantial level of Bdnf in control mice (Cnpcre:Bdnffl/+), whereas the O1+ or GFP+ fraction from Bdnf cKO mice showed significantly reduced level of Bdnf (Figure 5—figure supplement 2). Using presynaptic terminal recordings, we compared Bdnf cKO mice with control mice to examine how oligodendroglial BDNF affects presynaptic properties (Figure 5C).

Figure 5 with 2 supplements see all
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 a WT mouse (P10). (B) Conditional deletion of BDNF in oligodendrocytes (Cnpcre: Bdnffl/fl). Genotyping PCR using genomic DNA from control and Bdnf cKO mice, which are Cnpcre: Bdnffl/+ and Cnpcre: Bdnffl/fl, 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) Representative traces for membrane capacitance (Cm; top) and Ca2+ current (ICa; bottom) induced by 2-, 3-, 5-, 10-, and 20 ms depolarization (bottom) 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 ΔCm 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 Cm in WT and Bdnf cKO mice. (G) The plot of depolarization duration versus Ca2+ current charge (QICa) was generated from data as in (D) for both genotypes. (H) Left: Representative traces of ICa induced by a 100 ms step-like depolarization (from –80 to 60 mV, Δ10 mV) in control (black; n = 4) and Bdnf cKO mice (red; n = 5). Right: The I–V relationship for voltage-activated Ca2+ channels at the calyx terminal for each genotype is also shown. (I) Summary of the ΣΔCm 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).

https://doi.org/10.7554/eLife.42156.007

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, ΔCm 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 ΔCm and 40 ms- pulse exhibited saturation of ΔCm in both control and cKO calyces. ΔCm resulting from 2 ms depolarization was difficult to resolve in ~50% of Bdnf cKO calyces. In both genotypes, the Cm (15 ± 1.9 pF, n = 14 for control vs 15 ± 3.5 pF, n = 17 for Bdnf cKO; Figure 5F) and the Ca2+ influx during depolarizing pulses were similar (the QICa for 20 ms was 5.1 ± 0.45 pC/pF, n = 13 for the control vs 5.5 ± 0.39 pC/pF, n = 11 for the Bdnf cKO; p=0.45, unpaired t-test; Figure 5G), indicating that the loss of oligodendroglial BDNF impairs vesicular exocytosis without a change in Ca2+ channel activation, similar to what was observed in Bdnf+/– mice. In addition, the current–voltage relationship (I–V) curve for these voltage-activated Ca2+ 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 ΣΔCm 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 ΣΔCm 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 Pr 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 Pr (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.

Figure 6 with 1 supplement see all
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 (Pr, H). (I–J) Summary of the RRP size (I) and the Pr (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).

https://doi.org/10.7554/eLife.42156.010

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 μm3, n = 7 for control and 1199 ± 146 μm3, n = 6 for Bdnf cKO; p=0.3308, Mann-Whitney test; Figure 7—figure supplement 2). This result was consistent with the membrane capacitance (Cm) 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 (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.

Figure 7 with 2 supplements see all
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).

https://doi.org/10.7554/eLife.42156.012

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 control and Bdnf cKO mice (at P20) were prepared for EM imaging after 30 min pre-treatment with 7,8-DHF (20 μM) 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 glutamate vesicle release at presynaptic terminals in Bdnf cKO mice. The pre-application of BDNF (100 ng/ml) to brainstem slices for 30 min increased ΔCm in response to depolarizing pulses at the calyx terminal from Bdnf cKO mice. After 20 ms depolarizing pulses, ΔCm 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 QICa in treated and untreated terminals (for 20 ms pulses, 5.1 ± 0.47 pC/pF, n = 13 vs 5.8 ± 0.65 pC/pF, n = 5, respectively; p=0.42, unpaired t-test; Figure 8B). Interestingly, the application of BDNF had no effect on ΔCm and QICa in control calyces with a normal RRP (Figure 8—figure supplement 1). In addition, the direct activation of TrkB also rescued the impaired RRP and glutamate release at the calyx terminal in Bdnf cKO mice. After 20 ms depolarization pulses, ΔCm 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 QICa in the presence of 7,8-DHF (for 20 ms pulses, 5.1 ± 0.47 pC/pF, n = 13; p=0.42, unpaired t-test; Figure 8B). Furthermore, the ΣΔCm 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 ΣΔCm 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.

Figure 8 with 1 supplement see all
Application of extracellular BDNF or 7,8-DHF partially rescues the reduced exocytosis at calyx terminals in Bdnf cKO mice.

(A) Representative traces of Cm (top) and ICa (middle) induced by 20 ms depolarization from –80 to 0 mV (bottom) at calyx terminals in Bdnf cKO mice (P9–13, red) in the presence of BDNF (100 ng/ml; green) or 7,8-DHF (20 μM; blue). (B) The duration of depolarizing pulses was plotted versus ΔCm (left) and QICa (right) for terminals from Bdnf cKO slices in the absence (red) and the presence of BDNF (green) or 7,8-DHF (blue). (C) Representative traces of Cm (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 ΣΔCm relative to the stimulation time in the absence (red) or presence of 7,8-DHF (blue). (E) Summary of ΣΔCm 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).

https://doi.org/10.7554/eLife.42156.015

Oligodendroglial BDNF and auditory functions

To assess how loss of oligodendroglial BDNF and subsequent synaptic dysfunction influence auditory 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 peaks (herein referred to as waves I–V) during the 6 ms following a click stimulus and each wave corresponds to electrical responses from the auditory nerve (wave I) and the ascending auditory pathway (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 control and Bdnf cKO mice (42.8 ± 2.39 dB vs 42.8 ± 3.04 dB, n = 19 vs 14, respectively; Figure 9A,B). 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 significant difference in the amplitude of wave I, whereas the amplitudes of ABR waves II–IV were significantly 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 μV, n = 21 for control and 1.7 ± 0.16 μV, 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.

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).

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 neurotrophic 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 oligodendrocyte-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 impaired glutamatergic transmission without altering presynaptic Ca2+ channel activation. The results are comparable to a previous study in the inner ear showing the deletion of endogenous BDNF significantly reduced exocytosis of glutamate vesicles but did not affect Ca2+ 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 μM) 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 slowing down presynaptic Ca2+ 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 differentially 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). 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 Ca2+ channels, resulting in inhibition of Ca2+ channel activation rather than through BDNF-TrkB signaling. Further studies are required to determine the effect of exogenous BDNF on Ca2+ channel subtypes expressed in the presynaptic terminal and what aspects of BDNF signaling generate differential responses.

The mechanisms underlying the presynaptic effects of BDNF-TrkB signaling remain elusive. Activation 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 BDNF on the RRP could be mediated by the increases of intracellular Ca2+ levels, which may depend on the activation of the PLC pathway (Matsumoto et al., 2001; Reichardt, 2006). Recent studies demonstrated that BDNF-induced rise in intracellular Ca2+ concentration at the presynaptic terminal was mediated by Ca2+ influx through TRPC3 channels, resulting in a transient increase in spontaneous glutamate release (Cheng et al., 2017), and/or release of Ca2+ 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 functions 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 superior olive and the lateral superior olive (Wiechers et al., 1999; Hafidi, 1999). Glial cells may participate 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 present 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 brainstem during early postnatal development. Oligodendrocytes expressed BDNF in the MNTB of the auditory brainstem (Figure 1A), and isolated O1+ or Cnpcre-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-mediated signaling between oligodendrocytes and the calyx synapses induces BDNF release from oligodendrocytes 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 bidirectional neuron–glia communication at the calyx synapse through BDNF-dependent signaling during early postnatal development.

This study utilizes Cnpcre to generate a Bdnf deletion specifically in oligodendrocytes. A recent study reported that Cnpcre-driven YFP reporter signal was detected in 5.5% of NeuN+ neurons, suggesting a potential limitation on the specificity of recombination of Cnpcre mouse line (Tognatta et al., 2017). To address the specificity of Cnpcre in the MNTB, we have analyzed reporter 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 expressed Cnpcre-driven reporter in early postnatal ages (P10- P20). In addition, using FACS, isolated GCaMP6f-GFP+ cell population contains high levels of Olig2 mRNA with very low levels of Kcc2 mRNA, a neuronal marker (Figure 5—figure supplement 2). The majority (>95%) of Cnpcre 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 small percentage of neurons, affected by Cnpcre, 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 synapse has been observed to occur within a distance of 4.5 μm (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 pathway 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 mediated 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 speculate that bidirectional signaling occurs between oligodendrocytes and nerve terminals at synapses, in which glutamatergic inputs from neurons trigger oligodendrocytes to release BDNF, and then oligodendrocyte-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 represent an efficient way for oligodendrocytes to find active nerve terminals and to assist in maintaining 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
resource
DesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent
(M. musculus)
B6.129S4-Bdnftm1Jae/JThe Jackson Laboratorystock no.:002266PMID:8139657
Genetic reagent
(M. musculus)
Bdnftm3Jae/JThe Jackson Laboratorystock no.:004339Rios et al., 2001
Genetic reagent
(M. musculus)
CnpCre miceDr. Klaus Nave (Max Planck Institute)MGI no: 3051635Lappe-Siefke et al., 2003
Genetic reagent
(M. musculus)
GCaMP6fThe Jackson Laboratorystock no.:024105Dr. Paukert, UTHSCSA
Genetic reagent
(M. musculus)
tdTomatoThe Jackson Laboratorystock no.:007909Dr. Paukert, UTHSCSA
AntibodyMouse monoclonal anti-Olig1MilliporeMAB55401:500
AntibodyMouse monoclonal anti-MAP2MilliporeMAB34181:200
AntibodyMouse monoclonal anti-NeuNMilliporeMAB3771:200
AntibodyRabbit polyclonal anti-GFAPDAKOZ0334291:500
AntibodyMouse monoclonal anti-CC1MilliporeOP801:200
AntibodyRabbit monoclonal anti-Olig2Abcam1091861:100
AntibodyRat monoclonal anti-PDGFRaAbcamAB909671:300
AntibodyRabbit polyclonal anti-BDNFBiossBS4989R1:100
AntibodyMouse monoclonal anti-TrkBSanta Cruzsc-1369901:50
AntibodyGuinea pig
polyclonal anti-VGluT1
MilliporeAB59051:1000
Chemical compound, drug7,8-Dihydroxyflavone (7,8-DHF)SigmaD544620 μM
Chemical compound, drugBDNFMilliporeGF301100 ng/ml
Chemical compound, drugTEA-ClSigmaT226510 mM
Chemical compound, drug4-APSigma2758750.1 mM
Chemical compound, drugTTXTOCRIS10781 μM
Chemical
compound, drug
QX314 bromideTOCRIS10144 mM
Chemical compound, drugBicucullineTOCRIS13010 µM
Chemical compound, drugStrychnineSigmaS87532 µM

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 Committee protocols. BDNF heterozygous (Bdnf+/−) mice were generated by crossing Bdnf+/− mice (B6.129S4-Bdnftm1Jae/J; The Jackson Laboratory) with WT mice (C57B[L]6/J). The offspring were genotyped with a standard PCR assay. Primer sequences were as follows: forward, 5’-ATGCGTACCTGACTTTCTCCTTCT-3’; reverse, 5’-ACTGGGTGCTCAGGTACTGGTTGT-3’, which amplify a 280 bp and 350 bp fragment for Bdnf+/– mice and a 280 bp fragment for WT.

To create the cKO mice (Cnpcre: Bdnffl/fl), mice carrying the floxed allele of Bdnf (Bdnffl/fl; The Jackson Laboratory; Rios et al., 2001) were crossed to Cnpcre heterozygous mice (Lappe-Siefke et al., 2003). The constitutive KO allele is obtained after Cre-mediated recombination by crossing Cnpcre mice with Bdnffl/fl 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 analysis of tail genomic DNA using the appropriate primers: for Bdnffl/fl, forward, 5’-TGTGATTGTGTTTCTGGTGAC-3’ and reverse, 5’-GCCTTCATGCAACCGAAGTATG-3’, which amplifies a 487 bp (floxed Bdnf allele) and a 437 bp (Bdnf WT allele) fragment; for Cnpcre, forward, 5’-GCCACACATTCCTGCCCAAGCTC-3’ and reverse 1, 5’-GTCGCCACGCTGTCTTGGGCTCC-3’, and reverse 2, 5’-CTCCCACCGTCAGTACGTGAGAT-3’, which amplifies a 400 bp (Cnp WT allele) and 550 bp (Cnpcre allele) fragment. Control mice (Cnpcre: Bdnffl/+) were identified by PCR amplification of a 400 bp, 437 bp, and 487 bp fragment, whereas Bdnf cKO mice (Cnpcre: Bdnffl/fl) were identified by PCR amplification of a 400 bp, 550 bp, and 487 bp fragment. Recombination efficiency in oligodendrocytes in the Cnpcre 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

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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 MgCl2, 0.1 CaCl2, 25 glucose, 25 NaHCO3, 1.25 NaH2PO4, 0.4 ascorbic acid, three myoinositol, and 2 Na-pyruvate, pH 7.3–7.4 when bubbled with carbogen (95% O2, 5% CO2; osmolarity of 310–320 mOsm). Then, 200-µm-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 MgCl2 and 0.1 mM CaCl2 were replaced with 1 mM MgCl2 and 2 mM CaCl2.

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 software (HEKA Elektronik, Lambrecht/Pfalz, Germany). Slices were visualized using an infrared differential 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

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To measure presynaptic Ca2+ currents (ICa) and changes in membrane capacitance (ΔCm), the borosilicate glass pipettes were filled with a solution containing the following (in mM): 130 Cs-methanesulfonate, 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 MΩ. Series resistance was <20 MΩ before compensation and <10 MΩ with compensation. Presynaptic Ca2+ currents were analyzed after leak subtraction using a ‘traditional’ p/4 stimulus train in the EPC10-Patchmaster. For identification and morphological analyses, intracellular solutions were supplemented with 50 μM Alexa 568 (Life Technologies, USA). Extracellular aCSF solution contained 10 mM TEA-Cl, 0.1 mM 4-AP, and 1 μM TTX to block K+ and Na+ channels, respectively.

Postsynaptic recording

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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 bromide to block the voltage-activated Na+ current. Extracellular aCSF solution contained 10 µM bicuculline and 2 µM 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 MΩ. Series resistance was <20 MΩ, with 80% compensation. Afferent fibers of the calyx of Held synapses 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 constant 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

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Slices used for patch-clamp analysis or fresh brainstem slices (~200 µm 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; Millipore, 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

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Animals were anesthetized and intracardially perfused with normal saline. Brains were removed and 400-μm-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 Electron 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 recognizable 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 contacting the postsynaptic density. Docked and clustered vesicles were defined as those within 10 nm and 200 nm of the presynaptic active zone, respectively (Sätzler et al., 2002; Taschenberger et al., 2002).

ABR recordings

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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 O2 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 contralateral mastoid (ground). The electrical potential differences between the vertex and the mastoid electrodes 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-μs intervals. Acoustic stimuli were generated 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 duration) through TDT Multi-Field Magnetic Speakers placed 10 cm away from the left ear canal. A repetition 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 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)

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Bdnf cKO mice (CnpCre: Bdnffl/fl, n = 3) and control mice (CnpCre: Bdnffl/+, n = 2) were used for FACS experiments. CnpCre: Rosa-GCAMP6f-GFP+/- (n = 3) and CnpCre 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 μM, 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 μM). Mechanical titration was performed using a 1000 ul pipette tip followed by a 200 ul pipette tip. After dissociation, the suspension was filtered and spun down at 300x g for 7 min. The cells were resuspended in 400 μl of dissociation media. 50 μl were set aside for a no primary antibody control. 1 μl anti-O1 antibody (MAB1327, R and D systems) was added to the remaining 350 μl 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 μl 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 μl of dissociation solution. Sorting was performed on a BD FACSARIA III (BD Biosciences) in the Flow Cytometry Facility at UT Health San Antonio 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 doublet discrimination. 488-labeled O1+ cells were selected, yielding a population of 7,000–25,000 cells. In CnpCre: 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)

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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 Superscript III First-strand synthesis with 1 μl Oligo (dT) primers, 1 μl 10 mM dNTP mix (180180–051, Invitrogen). 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 μM. 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 (meanGene – meanGAPDH) was used to determine Delta Delta CT (Delta CTPos-Delta CTNeg) or (Delta CTcko-Delta CTCTL), relative gene expression was calculated 2^(-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 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 analyzed off-line and displayed with Igor Pro (Wavemetrics, Lake Oswego, OR). α values were set to 0.05, and all comparisons were two-tailed. To compare two groups, unpaired t-test or Mann-Whitney 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

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The authors declare that all data generated or analyzed in this study are available within the article.

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Decision letter

  1. Dwight E Bergles
    Reviewing Editor; Johns Hopkins University School of Medicine, United States
  2. Gary L Westbrook
    Senior Editor; Vollum Institute, United States
  3. Lu-Yang Wang
    Reviewer; Hospital for Sick Children, Canada

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "Oligodendrocytes regulate presynaptic properties and neurotransmission through BDNF signaling" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Lu-Yang Wang (Reviewer #2).

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

Summary:

All reviewers commented on the importance of understanding the interactions between oligodendrocytes and axons during development and the skill with which the physiological studies were carried out. The studies are clearly novel and interesting. However, as outlined below, major concerns were raised about the congruence between that ages of the mice subjected to the different manipulations and the veracity of the conclusions based on the analysis, in particular the voltage-dependence of Ca channels and the estimates of RRP. In addition, there is insufficient evidence provided that the genetic manipulations designed to delete BDNF from oligodendrocytes was selective and effective. Overall, these issues would have to be addressed before the study is considered sufficiently impactful to warrant publication in eLife.

Reviewer #1:

This manuscript from Kim and colleagues addresses an important question in developmental neuroscience – the role that myelinating glia play in shaping the maturation of neuronal excitability and synaptic connectivity. The authors exploit the extraordinary accessibility of the calyx synapse in the auditory brainstem to define how BDNF release from oligodendrocytes influences maturation of these synapses. The studies point to a role of this growth factor in altering the RRP, although the mechanism remains to be determined.

1) The authors generate CNPase-Cre; bdnffl/fl;Ai9 (tdTomato reporter) mice to achieve oligodendrocyte selective depletion of BDNF. It is crucial for the interpretation that they demonstrate both the selectivity and efficiency of gene deletion. Figure 5—figure supplement 1 evaluates co-localization between tdTomato and Olig1, which is used as a marker for oligodendrocytes. While there is some co-localization between Olig1 and tdTomato in panel A, many of the Olig1+ cells are not tdTomoto immunoreactive, and there appears to be widespread expression of tdTomato in vascular cells (perhaps pericytes). These panels are labeled as "CNPase", however they appear to show the distribution of tdTomato, as an indication of which cells experienced Cre dependent recombination. It is unusual to use Olig1 as a marker for oligodendrocyte cell bodies, rather than CNPase, CC1, or GSTPi, as Olig1 is expressed by other glial cells. The promiscuous expression of tdTomato in these mice raise concerns about specificity of the recombination, which is an issue, given the widespread expression of BDNF (see Figure 1). In addition, they have not provided evidence that this manipulation resulted in depletion of bdnf from oligodendrocytes. The standard experiment would be to isolate oligodendrocytes in this region by FACS and then perform qPCR to show that the mRNA is no longer detected. Immunolabeling for BDNF with the markers described above would unlikely to be sufficient, unless the co-localization were unambiguous.

2) The ABR measurements in Figure 8 indicate that there was a remarkable change in latency to the first peak in the bdnf cKO mice. This would suggest that conduction along the auditory nerve is slowed in these animals. This issue should be discussed, in light of the fact that CNPase-Cre will also induce recombination in peripheral Schwann cells.

3) There is no evaluation of whether loss of bdnf affects the density of oligodendrocyte progenitors or myelination (or Schwann cells). There are numerous reports that BDNF signaling through TrkB alters progenitor dynamics and the process of myelination. If myelination is altered, it could affect activity propagating along GBC axons and therefore maturation of their terminals. It is possible that there is sufficient BDNF coming from other sources to compensate, but if that is true, why don't these other sources compensate for the loss of oligodendrocyte-derived bdnf?

Reviewer #2:

By a combination of immunofluorescence labeling, patch-clamp electrophysiology, electron microscopy, and in vivo auditory brainstem response (ABR) tests in transgenic mice with conventional knockout (BDNF+/-) or oligodendrocyte-specific conditional knockout of BDNF (cKO), Jang et al. showed that oligodendrocyte derived BDNF is critical for boosting the size of the readily releasable pool (RRP) of synaptic vesicles (SVs) and actively promoting glutamate release from the calyx of Held terminals.

Overall this study presents compelling evidence to support the novel role of BDNF from oligodendrocytes to regulate synaptic transmission by targeting specific quantal parameter with advanced molecular and cellular tools. Conceptually, this study is important for the field of development plasticity in central synapses. This reviewer has no major objections to the main conclusion that the authors have drawn from their data but would like to make a few suggestions for the author to consider for tightening up the loose ends.

1) The authors framed the entire story that BDNF released specifically from oligodendrocytes are critical for modulating the RRP in the context of the critical period of development. However, the choice of age group for different experiments is quite confusing. For example, patch-clamp recordings of EPSCs were from P16-20 mice (mature or nearly mature synapses); immunofluorescence labeling and EM were done in P20-25 mice; and calcium currents and capacitance measurements were done in P9-13 mice (immature synapses). Given that dramatic changes in presynaptic spike waveform, calcium channel-SV coupling distance and morphological remodeling all occur during this period, it is difficult to discern if oligodendrocyte derived BDNF is specifically important for developmental remodeling of immature synapses and/or synaptic signaling in mature synapses. It would impart readers with more confidence if the authors can show the results from the experiments in parallel age groups.

2) With the entirely opposite results from Baydyuk et al., 2015, it is essential to show and validate the expression patterns of BDNF/TrkB for P9-13 age group in order to strengthen the arguments made in this paper against the previous paper.

3) In the capacitance jump experiments, it was stated that there is no difference in the basal membrane capacitance between WT and BDNF(+/-) calyces (Figure 3), but the calyx volume is significantly smaller in morphological analyses (Figure 6). There is an obvious conflict here which needs some explanation.

4) Given the abundant expression of TrkB in MNTB neurons, it is essential to analyze the effects of exogenous ligands on the amplitude and frequency of mEPSCs to ensure the observed effects is purely presynaptic as claimed (Figure 4). It is puzzling in fact that BDNF is not doing anything to postsynaptic neurons with much more abundant TrkB expression to detect BDNF than presynaptic terminals. It is probably too hasty to rule out the role of BDNF in postsynaptic signaling at this stage.

Reviewer #3:

The manuscript "Oligodendrocytes regulate presynaptic properties and neurotransmission through BDNF signaling", by Jian et al. examines the role of BDNF signaling on presynaptic function at the p8 -P25 mouse calyx of Held. Using the combination of mouse genetics, patch clamp electrophysiology, ultrastructural analysis, and tests of auditory function the authors make a novel and interesting finding that oligodendrocytes are a critical source of BDNF which is essential for proper synaptic transmission at the calyx of Held synapse. In addition, findings run counter to previous findings that BDNF is a negative regulator of synaptic transmission. In addition, their ability to carry out direct presynaptic recordings and measure calcium currents in combination with capacitance measurements allows for clear mechanistic interpretations of how BDNF regulation of synaptic transmission. Despite the novelty of the findings there are some issues that need to be resolved before acceptable for publication.

1) The mechanism by which BDNF rescues the RRP is unclear. The authors clearly show that with their capacitance measurements there is a reduction in the readily releasable pool at the calyx and that this can be rescued by application of BDNF. In addition, in the absence of BDNF their EM data clearly demonstrates a reduction in docked SV. Therefore, two possible interpretations can be made. Either each calyx contains the same number of active zones (AZs), but in the absence of each AZ has less docked SVs or absence of BDNF during development results in a reduction in the number of AZs. In both cases application of BDNF would result in an increase of docked SVs which would result in an increase on the RRP. The authors should show with EM that BDNF rescues the docking defect seen. In addition, the authors should carry out nonstationary EPSC variance analysis to determine the number of functional AZs in the presence and absence of BDNF.

2) The data concerning BDNF effects on the voltage dependence of activation on Calcium channels is not convincing. These measurements are highly dependent on the pipette series resistance in whole cell voltage clamp. There is no mention of the pipette series resistance during the actual recordings. Based on the example traces presented there appears to be differences in the tail current decay between WT and BDNF+/-. This could be due to differences in the quality of the voltage clamp or that reductions in BDNF do impact tail currents. In addition, it is not known if a leak subtraction protocol was performed. The authors should redo their IV analysis with better lower residual patch clamp pipette resistance. Furthermore, they should perform the IV analysis in 1.0 mM external Ca2+ to also offset potential issues with voltage clamp quality.

3) The authors demonstrate a reduction in AP evoked release and RRP as defined trains of APs at 100 Hz. It has been well established at the age groups p9-11 that a 3ms step depolarization corresponds to the RRP that can be released by action potentials. In Figure 5 which utilized p9-13, there appears to be no difference between the amount of exocytosis with a 2ms step pulse. Therefore, it is unclear why there is a dramatic effect in the AP RRP measurement and RRP as measured by the step depolarizations. It would be helpful if the authors did a 3ms Cm measurement to directly compare. Additionally, the authors should compare data collected at P9-11 vs P12-13 as mice begin to hear at P12. To accurate determine effects on BDNF regulation of the calyx RRP paired recordings at P9-11 would be ideal but not critical to their interpretations. Finally, why did they not carry out fiber stimulation at this age group too?

4) The calyx of Held goes through structural changes after hearing onset. The volumetric reconstruction of the P20-25 age group revealed significant reductions in calyx volume. However, the Capacitance measurements revealed no change in calyx volume as measured as pF. However, this comes from a different age group compared to the group in volume reconstructions. I am more inclined to trust the volumetric reconstructions since it is very difficult to accurately determine the calyx size with the patch clamp experiments. The authors should carry out volumetric reconstructions as the same age group or measure the calyx volume via a capacitance measurement in the P20-25 age group. Otherwise it is difficult to know why there is such a difference between the two measurements of calyx volume.

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for resubmitting your work entitled "Oligodendrocytes regulate presynaptic properties and neurotransmission through BDNF signalling" for further consideration at eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Gary Westbrook as the Senior Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below. In particular, there are remaining concerns about the specificity of recombination of the CNPase-Cre mice based on the information provided in the manuscript.

Reviewer #1:

Jang and colleagues have made substantial revisions to the manuscript and performed some very nice additional experiments to address concerns raised in the prior review. However, the additional data provided to address concerns about the specificity of recombination in CNPase mice are not sufficient.

1) In Figure 5—figure supplement 1, the authors show immunostaining for NeuN, GFAP, Pdgfra, CC1 and GFP to assess which cell types exhibit recombination. In these panels, GFP refers to GCaMP6f in CNPase-Cre x R26-lsl-GCaMP6f mice. There is diffuse GFP immunoreactivity throughout the GFP panels that is unaccounted for and unexpected if expression were restricted to oligodendrocytes. In addition, these images reveal intense regions of GFP immunoreactivity that co-localize with NeuN (cell in the center left of panel A) and co-localize with Pdgfra (panel C). Perhaps most significantly, in the GFP image of panel C there are several long processes and one cell body (center left) that is much larger than one would expect for an oligodendrocyte. For some reason, although this cell looks like a neuron, it also exhibits weak immunoreactivity to CC1. However, the CC1 immunoreactivity in panel C is very unusual, with several long processes visible (this is not normally observed with CC1 – see panel B) and many of the CC1+ cell bodies have unusual morphologies. Since orthogonal projections are not shown in this figure, it is difficult to assess whether labeling patterns show proximity or true co-localization. Thus, these immunostaining data to not provide strong support for the conclusion that CNPase-Cre mice exhibit selective recombination in oligodendrocytes. A minor point, in the figure legend, the mice are referred to as "CNPase-GCaMP6f-GFP mice". Please indicate the exact genotype of the mice for clarity.

2) The authors also perform PCR analysis on isolated cells to determine the specificity of recombination. However, no experimental details are provided for the FACS and PCR analysis shown in Figure 5. There is insufficient information provided that the isolation procedure was able to isolate a representative population of cells from the brainstem (astrocytes, neurons, oligos, etc.). Procedures used to preserve glia often result in widespread death of neurons, so it is important to assess what types of cells are in this pool.

3) The criteria used to determine positive versus negative O1 cells in Figure 5—figure supplement 2A is not indicated. From the second panel in A, it looks like there is a clear break between the blue/magenta cells and the green cells. However only the O1+ cells that exhibited the highest immunoreactivity were selected for the PCR analysis. This analysis should be repeated by including the blue cells, to provide a more representative sample of the population. It is unclear why GFP was not used to isolate cells in this example, as this would provide a more direct assessment of what types of cells experienced cre-dependent recombination. Because there is concern about neuronal recombination, neurons from this region should be isolated and GFP and BDNF expression assessed selectively in this pool.

4) The authors indicate that >27 cycles were necessary to detect BDNF message in the O1+ population. 27 cycles is typically used as a cutoff, with any product observed with more cycles being attributed to noise/non-specific amplification. This suggests that the amount of BDNF in O1+ cells is at the limit, or below the limit, of detection. Although more cycles were required to detect a product in the cKO O1+ cells, confidence in these data are low for this reason.

5) In Figure 5—figure supplement 2D immunostaining is performed in the cKO mice. The panels show that BDNF immunoreactivity is somewhat lower in O1+ cells in the cKO mice. As a constitutive cre was used, it is surprising that BDNF expression was not abolished. It also appears that BDNF immunoreactivity is also lower in neurons in the cKO mice. Thus, these data raise further questions about the specificity of recombination in these mice.

Reviewer #2:

The manuscript "Oligodendrocytes regulate presynaptic properties and neurotransmission through BDNF signaling" is a revision in response to previous comments. In this revision, they have performed new experiments and reanalyzed data. They have performed FACS and isolated oligodendrocytes to demonstrate that BDNF mRNA is knocked down specifically in oligodendrocytes in the BDNF cKO animal. They examined the loss of BDNF on calyx MNTB synaptic transmission and morphology at two developmental times point before hearing P10-12 calyces, and mature calyces P16-20. There data demonstrates that loss of BDNF impacts RRP size and release kinetics and presynaptic ultrastructure in a similar manner at both developmental time points. The authors have redone IV experiments and demonstrate that lack of BDNF does not impact calyx Ca2+ currents. Finally, they have analyzed RRP pool estimates using SMN plot in addition to their original EQ plots. These new data and analysis in addition to their previous novel findings on allows the authors to draw clear mechanistic conclusions on how oligodendrocytes BDNF signaling regulates presynaptic neurotransmitter release. These data are significant and represent a significant advancement in our understanding of synaptic plasticity. However, there are a few minor points in regards figures and data that the authors should correct and consider to improve the manuscript.

1) In general, for all bar graphs it would be ideal if the authors also provided a dot plot on top of the bar graphs. This would allow the reader to get an idea of the distribution of the date for the experiments.

2) In Figure 2 it would be ideal to also report the release probability measurements as determined by EQ plot measurement. Please report release probability measurements for EQ and SMN in Figure 6.

3) Figure 3 graphs appear to be missing the 3ms data point on all Panel B and Panel D. There are 5 measurements corresponding to the different depolarization times but only 4 data points on these graphs. In addition, in Panel F of the IV data it appears there is a shift in voltage dependence of activation. There is a lot of variance at the -40 mV point but the mean looks different. The authors should make a simple statement to address this potential issue as it could be due to differences in voltage clamp.

4) Figure 5 appears to be missing plotted IV data. In fact, I am confused by these traces. In the figure legend, it states traces showing 100 ms depolarizations from -80 mV to 0 mV. But there is no panel lettering to these traces.

5) Figure 7 is missing representative EM images from the immature calyx synapse. They need to be added.

6) Figure 7—figure supplement 1 is missing. Also, the volumetric data on the P20-25 calyces that was included in the previous version of the manuscript is now missing.

6) Figure 9. It would be helpful to delineate to the reader who are not auditory physiologist what the ABR waves correspond to.

Reviewer #3:

This revised version has significantly improved in its quality. The authors have addressed virtually all my criticisms. The action potential waveform is very important for the argument that RRP is specifically altered by BDNF from oligodendrocytes. I would suggest that stats for spike waveform in two age groups are included in the main figure or text.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Oligodendrocytes regulate presynaptic properties and neurotransmission through BDNF signalling" for further consideration at eLife. Your revised article has been favorably evaluated by Gary Westbrook as the Senior Editor and Dwight Bergles as the Reviewing Editor. The manuscript has been improved, but there are some remaining issues that need to be addressed before acceptance, as outlined below:

The final conclusion of the study is that oligodendrocytes in the brainstem release BDNF during development, which regulates the structural and functional maturation of calyceal synapses in the mouse MNTB. The effects on glutamate release are profound and well described through paired recordings and anatomical studies. The primary limitation of the study is that one mouse line, Cnp-Cre, was used to alter BDNF levels in oligodendrocytes. Because there is concern about the specificity of recombination in this mouse line, the study would be stronger if a subset of the effects were also assessed in a second oligodendrocyte specific Cre line. The authors did not include a reporter in the Cnp-Cre x Bdnfflox/flox cross to allow assessment of BDNF levels in the recombined cells and the selectivity of the recombination, instead relying on the surface antigen O1 for isolation of oligodendrocyte lineage cells for subsequent qPCR analysis. The authors show that samples enriched in oligodendrocyte lineage cells (O1+) have detectable bdnf mRNA by qPCR and that this level is reduced in the cKO mice. Although there is a trend towards lower bdnf mRNA in the O1- fraction, the value was not significantly lower (n=3). However, they did not specifically measure whether bdnf was lower in neurons (the O1- population presumably contains other cells, such as astrocytes which could mask any effects on neuron bdnf levels, as levels are typically higher in those cells). In addition, there is no quantification of BDNF protein in oligodendrocytes at this age, and no immuno-localization or in situ hybridization indicating the presence of BDNF protein or bdnf mRNA message in oligodendrocytes in this region. The authors find a small proportion of neurons in the brainstem exhibit evidence of recombination in this mouse line, and recombination outside the oligodendrocyte lineage (e.g. Schwann cells, immune cells) in Cnp-Cre mice has been described by others. This is significant, because BDNF levels are much higher in neurons than oligodendrocytes in cortical regions, although to our knowledge this hasn't been explored in the brainstem at this developmental stage. As BDNF is a secreted protein, any reduction in other cells could therefore contribute to the effects observed. At a minimum, the authors need to thoroughly address these limitations in the Discussion.

Additional concerns:

1) All data points should be added to the histograms, so that the readers can assess the distribution of values from individual experiments.

2) It would be helpful if the authors determined the identity of the tdTomato+ cells in Figure 5—figure supplement 1G by immunostaining.

3) There are numerous typographical errors that need to be corrected (e.g. Figure 5—figure supplement 1 – "Nuen"), the labels in some panels are unclear, and some formatting is off (e.g. Figure 5—figure supplement 2D). Throughout the study the authors used GCaMP6F as a reporter to show the specificity of recombination in the CNP-Cre line, but indicate "GFP" or GCaMP6f-GFP in the graphs and panels. This needs to be corrected throughout, for example, by indicating anti-GFP when indicating the antibody (rather than the protein) or GCaMP6F when indicating the protein.

4) The qPCR primer design should be changed so that the primers span across exons to reduce the possibility of genomic signal contamination.

5) The species should be indicated in the title.

https://doi.org/10.7554/eLife.42156.020

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

Summary:

All reviewers commented on the importance of understanding the interactions between oligodendrocytes and axons during development and the skill with which the physiological studies were carried out. The studies are clearly novel and interesting. However, as outlined below, major concerns were raised about the congruence between the ages of the mice subjected to the different manipulations.

We added new figures (new Figures 6 and 7 and supplementary figures) to address these concerns. We examine two different age groups from control and Bdnf cKO mice: Immature calyx synapses at P10-12 before hearing, and mature calyx synapse at P16-20 after hearing onset. At both time points, we examine presynaptic structural alterations with ultra-structural analysis using EM, and synaptic functions with EPSC recordings using whole-cell patch-clamp from each genotypes. At P10-12, before hearing onset, presynaptic recordings and EPSC recordings clearly demonstrate a reduction in vesicular neurotransmitter release in Bdnf cKO mice, and EM analysis shows that the number of docked vesicles is decreased in cKO mice. At P16-20, after hearing onset, EPSC recordings also show a reduction of vesicular neurotransmitter release in cKO mice, and EM analysis reveals that the number of docked vesicles is decreased in cKO mice. The EPSC and EM data are consistent between the two time points. Thus, we demonstrate that cKO continue to show altered synaptic function after hearing onset. These studies consistently show that OL-driven BDNF critically impacts mobilization of glutamate vesicles to the active zone to form the RRP during postnatal development.

And the veracity of the conclusions based on the analysis, in particular the voltage-dependence of Ca channels and the estimates of RRP.

As recommended in the reviewers’ comments, we re-analyze the voltage-dependence of Ca2+ channel currents and revise the I-V curve in the calyx terminals from WT, Bdnf+/-, control, and BDNF cKO mice using whole-cell voltage clamp with a lower series resistance (<20 MOhm before compensation and <10 MOhm after compensation) and analyze them after leak subtraction using HEKA EPC10 and Patchmaster. There are no differences in Ca2+ influx and voltage-dependence of Ca2+ channels between control and Bdnf cKO, or between WT and Bdnf+/- mice. We added the newly analyzed data and representative traces in Figures 3 and 5.

In addition, we added the results using the Schneggenburger-Meyer-Neher (SMN) method to estimate the RRP size and the replenishment rate of vesicles from WT, Bdnf+/−, control, and Bdnf cKO. New figures and data were added in Figure 2 and Figure 6—figure supplement 1. These additional studies further strengthen the data supporting alterations in the RRP due to the reduction of BDNF and OL-derived BDNF, specifically.

In addition, there is insufficient evidence provided that the genetic manipulations designed to delete BDNF from oligodendrocytes was selective and effective.

We added new data to confirm that Cre-dependent recombination in CnCre mice is specific to OLs. We show immunostaining for CC1, PDGFRα, and Olig2 as OL markers, and verify their colocalization with the Cre-dependent reporter, GCaMP6f (Figure 5—figure supplement 1). In addition, we perform FACS to isolate O1+ OLs and utilize qPCR to show the level of BDNF mRNA in OLs, as reviewer’s suggested. BDNF RNA is detected in the O1+ OLs isolated from control mice. Importantly, BDNF levels in the O1+ fraction of Bdnf cKO mice were significantly reduced compared to control mice. There was no difference in the cKO O1- fraction compared to the control O1- fraction. The results indicate that the knockdown of BDNF occurs specifically in OLs in cKO mice. We added this data in Figure 5—figure supplement 2.

Overall, these issues would have to be addressed before the study is considered sufficiently impactful to warrant publication in eLife.

Reviewer #1:

[…] 1) The authors generate CNPase-Cre; bdnffl/fl;Ai9 (tdTomato reporter) mice to achieve oligodendrocyte selective depletion of BDNF. It is crucial for the interpretation that they demonstrate both the selectivity and efficiency of gene deletion. Figure 5—figure supplement 1 evaluates co-localization between tdTomato and Olig1, which is used as a marker for oligodendrocytes. While there is some co-localization between Olig1 and tdTomato in panel A, many of the Olig1+ cells are not tdTomoto immunoreactive, and there appears to be widespread expression of tdTomato in vascular cells (perhaps pericytes). These panels are labeled as "CNPase", however they appear to show the distribution of tdTomato, as an indication of which cells experienced Cre dependent recombination. It is unusual to use Olig1 as a marker for oligodendrocyte cell bodies, rather than CNPase, CC1, or GSTPi, as Olig1 is expressed by other glial cells. The promiscuous expression of tdTomato in these mice raise concerns about specificity of the recombination, which is an issue, given the widespread expression of BDNF (see Figure 1). In addition, they have not provided evidence that this manipulation resulted in depletion of bdnf from oligodendrocytes. The standard experiment would be to isolate oligodendrocytes in this region by FACS and then perform qPCR to show that the mRNA is no longer detected. Immunolabeling for BDNF with the markers described above would unlikely to be sufficient, unless the co-localization were unambiguous.

As mentioned above, we added new data to confirm that Cre-dependent recombination in CNPCre mice is specific to OLs (Figure 5—figure supplement 1). In addition, we perform FACS to isolate O1+ OLs and utilize qPCR to show the level of BDNF mRNA in OLs, as reviewer’s suggested. O1 is a commonly used marker for OLs and recognizes galactocerebroside. O1 recognizes an external epitope that enables FACS with live stained cells. To validate that we successfully isolated OLs, we compared the relative gene expression of Olig2 in the O1- and O1+ fraction from control mice. The O1+ fraction has Olig2 levels that were 6.73x greater than the O1- fraction (O1- = 100.1 ± 5.2, O1+ = 673.3 ± 17.6, p<0.0001, n = 3 mice). BDNF RNA is detected in the O1+ OLs isolated from control mice (mean cycle threshold (CT) = 27.8 ± 0.08, n = 3 mice). Importantly, BDNF levels in the O1+ fraction of Bdnf cKO mice are significantly reduced compared to control mice (Control = 33.4 ± 8.4 vs. cKO = 14.67 ± 0.6, p = 0.0054, n = 3 mice/genotype). There is no difference in the cKO O1- fraction compared to the control O1- fraction (Control = 100.3 ± 7.5 vs. cKO = 89.3 ± 11.8, p = 0.47, n = 3 mice/genotype). The results indicate that the knockdown of BDNF occurs specifically in OLs. We added this data in Figure 5—figure supplement 2.

“To study the specific role of oligodendrocytes in presynaptic functions as BDNF providers, we generated Bdnf cKO mice, in which BDNF was specifically deleted in CNPase-expressing (CNPase+) oligodendrocytes using the Cre/loxP system (Figure 5B). […] Using presynaptic terminal recordings, we compared Bdnf cKO mice with control mice (Bdnffl/+ CNPCre–/–) to examine how oligodendroglial BDNF affects presynaptic properties (Figure 5C).”

2) The ABR measurements in Figure 8 indicate that there was a remarkable change in latency to the first peak in the bdnf cKO mice. This would suggest that conduction along the auditory nerve is slowed in these animals. This issue should be discussed, in light of the fact that CNPase-Cre will also induce recombination in peripheral Schwann cells.

There is no significant difference in the latency of the first ABR wave from Bdnf cKO mice, indicating no significant change in peripheral conduction. We also analyzed the central conduction time, defined as the time difference between ABR wave II and wave IV. There is no significant difference in the latency of ABR waves, indicating that peripheral and central conduction time are not altered in Bdnf cKO. We added the data for peripheral and central conduction to the manuscript (Figure 9).

3) There is no evaluation of whether loss of bdnf affects the density of oligodendrocyte progenitors or myelination (or Schwann cells). There are numerous reports that BDNF signaling through TrkB alters progenitor dynamics and the process of myelination. If myelination is altered, it could affect activity propagating along GBC axons and therefore maturation of their terminals. It is possible that there is sufficient BDNF coming from other sources to compensate, but if that is true, why don't these other sources compensate for the loss of oligodendrocyte-derived bdnf?

This manuscript addresses the role of OL-driven BDNF in synaptic transmission. We are studying the effect of BDNF on myelination in the auditory nervous system in another project. We examined axon myelination in BDNF cKO mice using electron microscopy to evaluate whether loss of OL BNDF impacts myelination, and found that there is no significant difference in axon diameter and myelin thickness by analyzing g-ratio.

Reviewer #2:

[…] 1) The authors framed the entire story that BDNF released specifically from oligodendrocytes are critical for modulating the RRP in the context of the critical period of development. However, the choice of age group for different experiments is quite confusing. For example, patch-clamp recordings of EPSCs were from P16-20 mice (mature or nearly mature synapses); immunofluorescence labeling and EM were done in P20-25 mice; and calcium currents and capacitance measurements were done in P9-13 mice (immature synapses). Given that dramatic changes in presynaptic spike waveform, calcium channel-SV coupling distance and morphological remodeling all occur during this period, it is difficult to discern if oligodendrocyte derived BDNF is specifically important for developmental remodeling of immature synapses and/or synaptic signaling in mature synapses. It would impart readers with more confidence if the authors can show the results from the experiments in parallel age groups.

We appreciate the reviewer’s constructive comments. To address the concerns about the influence of age, we added new data for two different time points in control and Bdnf cKO. We compared the data from postsynaptic EPSC recordings, presynaptic capacitance measurements, and EM in the immature calyx synapse (at P10-P12), and we demonstrated EPSC recordings and EM at the mature calyx synapse (at P16-P20) in parallel. The data from both EPSC recordings and EM indicate a sustained effect of reduced OL BDNF at P16-P20. Taken together, OL-derived BDNF regulates presynaptic RRP in both the immature and mature synapses. We added new data in Figure 6 and Figure 6—figure supplement 1.

2) With the entirely opposite results from Baydyuk et al., 2015, it is essential to show and validate the expression patterns of BDNF/TrkB for P9-13 age group in order to strengthen the arguments made in this paper against the previous paper.

The conflicting findings may result from the differences between species, ages, or BDNF application method. In Baydyuk et al., 2015, the authors show BNDF and TrkB expression in calyces using BDNF and TrkB knock-in mouse strains, but BDNF and TrkB expression were not shown in WT animals. However, all electrophysiological experiments had been done in the brainstem of Wistar rats. Thus BDNF effects could be influenced by species or strain of experimental animals. Another possibility is the BDNF application method influences the results as acute exposure likely differs from in vivo knockout. In our current study, we detected TrkB signals from calyces in both control and cKO mice in the same tissue used for electrophysiology. BDNF expression was detected in OLs as well as MNTB neurons in control, whereas OL BDNF expression was significantly reduced in cKO mice. We added BDNF and TrkB immunostaining in the MNTB from control and cKO mice to the manuscript (Figure 5—figure supplement 2 and Figure 4).

3) In the capacitance jump experiments, it was stated that there is no difference in the basal membrane capacitance between WT and BDNF(+/-) calyces (Figure 3), but the calyx volume is significantly smaller in morphological analyses (Figure 6). There is an obvious conflict here which needs some explanation.

In both the Bdnf+/- and cKO mice, we found no difference in the basal membrane capacitance in electrophysiological recordings (20 ± 1.5 pF, n = 10 for WT vs 21.5 ± 1.35 pF, n = 9 for Bdnf+/-, P = 0.43; Figure 3C, and 15 ± 1.9 pF, n=14 for control vs 15 ± 3.5 pF, n=17 for Bdnf cKO, Figure 5). In Bdnf cKO mice (at P10-12), we patched calyces and filled Alexa 568. After taking confocal images, we did volumetric 3D-reconstruction of calyx images and analyzed their volume using Amira software. We found that the volume has a tendency toward a slight reduction in the Bdnf cKO, but it was not significantly different from control. The volume of the calyx terminal was not significantly different in Bdnf cKO mice (1,378 ± 143.7 mm3, n = 7 for control and 1,199 ± 146 mm3, n = 6 for Bdnf cKO;P = 0.3308, Figure 7—figure supplement 1). We revised the manuscript and the figure to reflect this finding.

4) Given the abundant expression of TrkB in MNTB neurons, it is essential to analyze the effects of exogenous ligands on the amplitude and frequency of mEPSCs to ensure the observed effects is purely presynaptic as claimed (Figure 4). It is puzzling in fact that BDNF is not doing anything to postsynaptic neurons with much more abundant TrkB expression to detect BDNF than presynaptic terminals. It is probably too hasty to rule out the role of BDNF in postsynaptic signaling at this stage.

It is well known that BDNF regulates neuronal survival and growth in the developing brain. Thus BDNF could be involved in postsynaptic modulation and cellular signaling. In this study, we focus on studying the role of oligodendroglial BDNF in presynaptic properties and neurotransmitter release. In our exclusive experimental design using the Bdnf+/- or cKO mice, we did not observe postsynaptic alterations although we could not rule out the role of BDNF in postsynaptic signaling.

Reviewer #3:

[…] 1) The mechanism by which BDNF rescues the RRP is unclear. The authors clearly show that with their capacitance measurements there is a reduction in the readily releasable pool at the calyx and that this can be rescued by application of BDNF. In addition, in the absence of BDNF their EM data clearly demonstrates a reduction in docked SV. Therefore, two possible interpretations can be made. Either each calyx contains the same number of active zones (AZs), but in the absence of each AZ has less docked SVs or absence of BDNF during development results in a reduction in the number of AZs. In both cases application of BDNF would result in an increase of docked SVs which would result in an increase on the RRP. The authors should show with EM that BDNF rescues the docking defect seen. In addition, the authors should carry out nonstationary EPSC variance analysis to determine the number of functional AZs in the presence and absence of BDNF.

As it was mentioned above, we added new data for the rescue of the docking deficit using EM, which show that the TrkB agonist (7,8-DHF) rescues the deficit in docked vesicles at the calyx terminal shown in cKO mice (Figure 7). We did not include the number of AZs in this manuscript, and mentioned the possibility of changes in the number of AZs in the Discussion.

2) The data concerning BDNF effects on the voltage dependence of activation on Calcium channels is not convincing. These measurements are highly dependent on the pipette series resistance in whole cell voltage clamp. There is no mention of the pipette series resistance during the actual recordings. Based on the example traces presented there appears to be differences in the tail current decay between WT and BDNF+/-. This could be due to differences in the quality of the voltage clamp or that reductions in BDNF do impact tail currents. In addition, it is not known if a leak subtraction protocol was performed. The authors should redo their IV analysis with better lower residual patch clamp pipette resistance. Furthermore, they should perform the IV analysis in 1.0 mM external Ca2+ to also offset potential issues with voltage clamp quality.

In the revision, we re-examined the voltage-dependence of Ca2+ channel currents in the calyx terminals from WT, Bdnf+/-, control, and Bdnf cKO in whole-cell voltage clamp with a lower series resistance and re-analyzed them after leak subtraction using the “traditional” p/4 stimulus train in EPC10-Patchmaster. We addressed the series resistance during the recordings in the Materials and methods; the series resistance was 15 ± 3.1 MOhm before compensation (n=10), adjusted to <10 MOhm after compensation of ~ 50%. When the patch recordings maintain a lower series resistance to avoid potential issues with voltage clamp quality, there is no difference in the voltage dependence of Ca2+ channels between WT and Bdnf+/- mice. The I-V curve was newly updated in Figure 3. We added the newly analyzed data and representative traces in Figures 3 and 5.

3) The authors demonstrate a reduction in AP evoked release and RRP as defined trains of APs at 100 Hz. It has been well established at the age groups p9-11 that a 3ms step depolarization corresponds to the RRP that can be released by action potentials. In Figure 5 which utilized p9-13, there appears to be no difference between the amount of exocytosis with a 2ms step pulse. Therefore, it is unclear why there is a dramatic effect in the AP RRP measurement and RRP as measured by the step depolarizations. It would be helpful if the authors did a 3ms Cm measurement to directly compare. Additionally, the authors should compare data collected at P9-11 vs P12-13 as mice begin to hear at P12. To accurate determine effects on BDNF regulation of the calyx RRP paired recordings at P9-11 would be ideal but not critical to their interpretations. Finally, why did they not carry out fiber stimulation at this age group too?

We added data with the 3 ms step pulse at P9-12 in Figure 5. In control, 2- and 3-ms depolarizing pulses induced a capacitance jump (ΔCm) of 51 ± 8.6 fF and 72 ± 16.5 fF, n=12, respectively, which are comparable to those in age-matched mouse calyces, previously describe in Lin and Taschenberger, 2011. In Bdnf cKO calyces, ΔCm in response to 2-ms depolarization was difficult to resolve, and 3-ms depolarization induced a ΔCm of 27 ± 10.5 fF, n=10. A longer depolarization induced a larger ΔCm, and pulse durations > 40 ms exhibited saturation of ΔCm in both control and cKO calyces (Figure 5E, Lin et al., 2011).

For the fiber stimulation, we added new data for evoked EPSCs in immature (P10-12, before hearing onset) and mature (P16-20, after hearing onset, Figure 6) MNTB neurons from control and cKO mice.

4) The calyx of Held goes through structural changes after hearing onset. The volumetric reconstruction of the P20-25 age group revealed significant reductions in calyx volume. However, the Capacitance measurements revealed no change in calyx volume as measured as pF. However, this comes from a different age group compared to the group in volume reconstructions. I am more inclined to trust the volumetric reconstructions since it is very difficult to accurately determine the calyx size with the patch clamp experiments. The authors should carry out volumetric reconstructions as the same age group or measure the calyx volume via a capacitance measurement in the P20-25 age group. Otherwise it is difficult to know why there is such a difference between the two measurements of calyx volume.

Thanks for the reviewer’s thoughtful comments. The volumetric reconstruction from P10-12 calyces after presynaptic capacitance measurement was reanalyzed. There was no difference in the membrane capacitance and the volume of the calyx from 3D reconstruction between control and cKO (at P10-12). The volume of the calyx terminal was not significantly different in Bdnf cKO mice (1,378 ± 143.7 mm3, n = 7 for control and 1,199 ± 146 mm3, n = 6 for Bdnf cKO;P = 0.3308, Figure 7—figure supplement 1). This result was comparable to their membrane capacitance (Cm) measurement; there was no difference (15 ± 1.9 pF, n=14 for control vs. 15 ± 3.5 pF, n=17 for Bdnf cKO, Figure 5). We added this new data in Figure 7—figure supplement 1.

Author response image 1
(Left) 3D images of calyces from control and cKO mice.

(Right) Summary of volume and Cm.

[Editors' note: the author responses to the re-review follow.]

Reviewer #1:

Jang and colleagues have made substantial revisions to the manuscript and performed some very nice additional experiments to address concerns raised in the prior review. However, the additional data provided to address concerns about the specificity of recombination in CNPase mice are not sufficient.

1) In Figure 5—figure supplement 1, the authors show immunostaining for NeuN, GFAP, Pdgfra, CC1 and GFP to assess which cell types exhibit recombination. In these panels, GFP refers to GCaMP6f in CNPase-Cre x R26-lsl-GCaMP6f mice. There is diffuse GFP immunoreactivity throughout the GFP panels that is unaccounted for and unexpected if expression were restricted to oligodendrocytes. In addition, these images reveal intense regions of GFP immunoreactivity that co-localize with NeuN (cell in the center left of panel A) and co-localize with Pdgfra (panel C). Perhaps most significantly, in the GFP image of panel C there are several long processes and one cell body (center left) that is much larger than one would expect for an oligodendrocyte. For some reason, although this cell looks like a neuron, it also exhibits weak immunoreactivity to CC1. However, the CC1 immunoreactivity in panel C is very unusual, with several long processes visible (this is not normally observed with CC1 – see panel B) and many of the CC1+ cell bodies have unusual morphologies. Since orthogonal projections are not shown in this figure, it is difficult to assess whether labeling patterns show proximity or true co-localization. Thus, these immunostaining data to not provide strong support for the conclusion that CNPase-Cre mice exhibit selective recombination in oligodendrocytes. A minor point, in the figure legend, the mice are referred to as "CNPase-GCaMP6f-GFP mice". Please indicate the exact genotype of the mice for clarity.

To address the concerns about the immunohistochemical analysis we have added a new representative image with orthogonal views orthogonal views to aid in the visualization of the colocalized cells. In addition we added quantitative analysis of co-localization using two different neuronal markers (NeuN and Map2) and two reporter lines (GCaMP6f and tdTomato reporter lines). The quantitative analysis demonstrates that >97% of the recombined cells are OLs with a very small portion of neurons showing positive staining (< 3%, See the Figure 5—figure supplement 1).

2) The authors also perform PCR analysis on isolated cells to determine the specificity of recombination. However, no experimental details are provided for the FACS and PCR analysis shown in Figure 5. There is insufficient information provided that the isolation procedure was able to isolate a representative population of cells from the brainstem (astrocytes, neurons, oligos, etc.). Procedures used to preserve glia often result in widespread death of neurons, so it is important to assess what types of cells are in this pool.

We have added additional qPCR analysis to include the neuronal marker, Kcc2. There is no reduction of the Kcc2 in sorted neuronal populations. Experimental details have been added to the Materials and methods section. We carefully verified the isolated cells. Isolated cells from Cnpcre+/-; GCAMP6f-GFP+ cells versus GFP- cells demonstrated an enrichment of Olig2 RNA quantified by qPCR. Kcc2, was depleted from the GFP+ population, indicating that GFP+ cells are Olig2+ and Kcc2 negative OLs (see the Figure 5—figure supplement 2).

3) The criteria used to determine positive versus negative O1 cells in Figure 5—figure supplement 2A is not indicated. From the second panel in A, it looks like there is a clear break between the blue/magenta cells and the green cells. However only the O1+ cells that exhibited the highest immunoreactivity were selected for the PCR analysis. This analysis should be repeated by including the blue cells, to provide a more representative sample of the population. It is unclear why GFP was not used to isolate cells in this example, as this would provide a more direct assessment of what types of cells experienced cre-dependent recombination. Because there is concern about neuronal recombination, neurons from this region should be isolated and GFP and BDNF expression assessed selectively in this pool.

The selection of the positive population in FACS was performed using scatter, histogram analysis, and the expertise of the FACS CORE. We have added data from Cnpcre: R26-lsl-GCaMP6f mice that demonstrate an enrichment of Olig2 in the GFP+ cells and an absence of Kcc2. We have not yet crossed these mice with the Bdnf flox/flox, thus do not provide data from those mice.

4) The authors indicate that >27 cycles were necessary to detect BDNF message in the O1+ population. 27 cycles is typically used as a cutoff, with any product observed with more cycles being attributed to noise/non-specific amplification. This suggests that the amount of BDNF in O1+ cells is at the limit, or below the limit, of detection. Although more cycles were required to detect a product in the cKO O1+ cells, confidence in these data are low for this reason.

We demonstrate that the Bdnf primers detect a reduction in Bdnf RNA and thus are specific using Bdnf heterozygous mice compared to littermate controls. We show that the Bdnf primers linearly amplify even with low amounts of input RNA and cycles >27, indicating that the Bdnf primers can detect differences in RNA within the CT range observed in these studies (see the Materials and methods section).

5) In Figure 5—figure supplement 2D immunostaining is performed in the cKO mice. The panels show that BDNF immunoreactivity is somewhat lower in O1+ cells in the cKO mice. As a constitutive cre was used, it is surprising that BDNF expression was not abolished. It also appears that BDNF immunoreactivity is also lower in neurons in the cKO mice. Thus, these data raise further questions about the specificity of recombination in these mice.

Due to concerns about the specificity of the BDNF antibody, we have removed the immunostaining. As we have shown with immunohistochemistry, the Cnpcre targets a subpopulation of OLs. Thus, the presence of BDNF in the O1+ from Bdnf cKO mice likely comes from Cnpcre-/- O1+ OLs.

Reviewer #2:

1) In general, for all bar graphs it would be ideal if the authors also provided a dot plot on top of the bar graphs. This would allow the reader to get an idea of the distribution of the date for the experiments.

We did not change the bar graphs to the dot plot on the bar graphs, saving our efforts on the remaking all figures. Instead, we focused on adding more data to complete the manuscript following reviewer’s comments. All data set will be shared, thus if anyone wants to look the distribution of the data, it will be available to access the data set.

2) In Figure 2 it would be ideal to also report the release probability measurements as determined by EQ plot measurement. Please report release probability measurements for EQ and SMN in Figure 6.

We did. The release probability measurements as determined by EQ plot measurement was added in the main text (Figure 2). Release probability measurements for EQ and SMN were reported in Figure 6.

3) Figure 3 graphs appear to be missing the 3ms data point on all Panel B and Panel D. There are 5 measurements corresponding to the different depolarization times but only 4 data points on these graphs. In addition, in Panel F of the IV data it appears there is a shift in voltage dependence of activation. There is a lot of variance at the -40 mV point but the mean looks different. The authors should make a simple statement to address this potential issue as it could be due to differences in voltage clamp.

We added the missing points for 3 ms in Figure 3B and D. We added more data for the IV curve in Figure 3F, showing no difference the mean value at the -40 mV.

4) Figure 5 appears to be missing plotted IV data. In fact, I am confused by these traces. In the figure legend, it states traces showing 100 ms depolarizations from -80 mV to 0 mV. But there is no panel lettering to these traces.

We added the IV curve of Ca2+ current from control and cKO in Figure 5. The figure legend was revised.

5) Figure 7 is missing representative EM images from the immature calyx synapse. They need to be added.

We added representative EM image from the immature calyx synapse to Figure 7—figure supplement 1.

6) Figure 7—figure supplement 1 is missing. Also, the volumetric data on the P20-25 calyces that was included in the previous version of the manuscript is now missing.

We added Figure 7—figure supplement 2, including the volumetric data.

6) Figure 9. It would be helpful to delineate to the reader who are not auditory physiologist what the ABR waves correspond to.

We did.

Reviewer #3:

This revised version has significantly improved in its quality. The authors have addressed virtually all my criticisms. The action potential waveform is very important for the argument that RRP is specifically altered by BDNF from oligodendrocytes. I would suggest that stats for spike waveform in two age groups are included in the main figure or text.

We added the statistic data for the amplitude and half-width of spike waveform from WT and hetero calyces to the Results section.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Oligodendrocytes regulate presynaptic properties and neurotransmission through BDNF signalling" for further consideration at eLife. Your revised article has been favorably evaluated by Gary Westbrook as the Senior Editor, and Dwight Bergles as the Reviewing Editor. The manuscript has been improved, but there are some remaining issues that need to be addressed before acceptance, as outlined below:

The final conclusion of the study is that oligodendrocytes in the brainstem release BDNF during development, which regulates the structural and functional maturation of calyceal synapses in the mouse MNTB. The effects on glutamate release are profound and well described through paired recordings and anatomical studies. The primary limitation of the study is that one mouse line, Cnp-Cre, was used to alter BDNF levels in oligodendrocytes. Because there is concern about the specificity of recombination in this mouse line, the study would be stronger if a subset of the effects were also assessed in a second oligodendrocyte specific Cre line.

We recently established a second Bdnf cKO mouse (Sox10-creER: Bdnff/f) using a second oligodendrocyte-specific Cre line (Sox10-creER mice, provided by Dr. Shin Kang, Temple University). In this second cKO mouse, we found the same phenotype of presynaptic terminals in the calyx of Held synapse with those described in the manuscript (see Author response image 2). We are preparing a separate manuscript using this mouse line, thus the following data set is not included in the current manuscript.

Author response image 2
Effects of reduced BDNF in OL on vesicular glutamate release from presynaptic terminal in the Bdnf cKO (Sox10creER: Bdnff/f).

(A) Representative traces for membrane capacitance (Cm; top) and Ca2+ current (ICa; bottom) induced by 10‐ms depolarization from P11–12 calyx terminals in control (black) and Bdnf cKO (red) mice. (B‐C) Summary of Ica and Cm induced by 10‐ms depolarization to 0 mV in calyces from control (black) and cKO (red) mice. (D) Depolarization duration plotted against ΔCm for control and Bdnf cKO mice. (E) Verification of Cre-line specificity. Using FACS and qPCR, isolated GCaMP6f‐GFP+ cells demonstrated a high level of Olig2 mRNA, a oligodendrocytes marker, but no detectable Kcc2 mRNA, a neuronal marker, indicating this Cre‐line is specific to Olig2+ oligodendrocytes.

The authors did not include a reporter in the Cnp-Cre x Bdnfflox/flox cross to allow assessment of BDNF levels in the recombined cells and the selectivity of the recombination, instead relying on the surface antigen O1 for isolation of oligodendrocyte lineage cells for subsequent qPCR analysis. The authors show that samples enriched in oligodendrocyte lineage cells (O1+) have detectable bdnf mRNA by qPCR and that this level is reduced in the cKO mice. Although there is a trend towards lower bdnf mRNA in the O1- fraction, the value was not significantly lower (n=3). However, they did not specifically measure whether bdnf was lower in neurons (the O1- population presumably contains other cells, such as astrocytes which could mask any effects on neuron bdnf levels, as levels are typically higher in those cells).

We added new data for the assessment of Bdnf mRNA levels in the recombined cells and selectivity of the recombination using the Cnpcre:Bdnf f/f;GCaMP6f-GFP mice (n=3 animals). The result indicated 1) the presence of Bdnf mRNA in OLs in the MNTB of the brainstem from control, and 2) the knock-down of Bdnf mRNA expression in OLs in Bdnf cKO mice (Figure 5—figure supplement 2).

In addition, there is no quantification of BDNF protein in oligodendrocytes at this age, and no immuno-localization or in situ hybridization indicating the presence of BDNF protein or bdnf mRNA message in oligodendrocytes in this region. The authors find a small proportion of neurons in the brainstem exhibit evidence of recombination in this mouse line, and recombination outside the oligodendrocyte lineage (e.g. Schwann cells, immune cells) in Cnp-Cre mice has been described by others. This is significant, because BDNF levels are much higher in neurons than oligodendrocytes in cortical regions, although to our knowledge this hasn't been explored in the brainstem at this developmental stage. As BDNF is a secreted protein, any reduction in other cells could therefore contribute to the effects observed.

Our results showed the presence of BDNF in oligodendrocytes in the MNTB of the mouse brainstem during early postnatal development. ~ 50% of Olig1+ oligodendrocytes were positive for BDNF immunostaining (Figure 1A). Using FACS and qPCR, we demonstrated that O1+ cells have detectable Bdnf mRNA, which was significantly reduced in Bdnf cKO mice. In addition, GCaMP6f-GFP+ cells also showed a substantial amount of Bdnf mRNA in control, which was significantly reduced in Bdnf cKO mice (newly added to Figure 5—figure supplement 2). There was no significant difference in the level of Bdnf mRNA in the O1− fraction or GFP- fraction in cKO versus control, although there was a trend towards lower Bdnf mRNA. The sorting likely did not completely isolate the oligodendrocytes and the remaining population could contribute to the slight reduction in Bdnf mRNA. Another possibility is the small percentage of neurons that are affected is contributing to this trend. However, it is unlikely that BDNF reduction in <5% neurons have a global and major impact on the synaptic function in the MNTB. Due to the biochemical nature of BDNF, it is thought to act locally at the synapse with limited diffusion (Sasi, 2017). The effect of BDNF within a synapse has been observed to occur with the low micrometer range (within a distance of 4.5 µm, Horch, 2002). Thus, BDNF reduction in the small portion of neurons (<5%) may not critically contribute to 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. As we addressed above, studies using additional oligodendroglia cre line such as Sox10-CreER mice confirm this interpretation.

At a minimum, the authors need to thoroughly address these limitations in the Discussion.

We rewrote the Discussion including the issue about the Cre line raised by the reviewer and editor.

Additional concerns:

1) All data points should be added to the histograms, so that the readers can assess the distribution of values from individual experiments.

We changed all bar graphs to include all data points.

2) It would be helpful if the authors determined the identity of the tdTomato+ cells in Figure 5—figure supplement 1G by immunostaining.

We added the panel showing the identity of the tdTomato+ cells, which are positive for Olig2.

3) There are numerous typographical errors that need to be corrected (e.g. Figure 5—figure supplement 1 – "Nuen"), the labels in some panels are unclear, and some formatting is off (e.g. Figure 5—figure supplement 2D). Throughout the study the authors used GCaMP6F as a reporter to show the specificity of recombination in the CNP-Cre line, but indicate "GFP" or GCaMP6f-GFP in the graphs and panels. This needs to be corrected throughout, for example, by indicating anti-GFP when indicating the antibody (rather than the protein) or GCaMP6F when indicating the protein.

We corrected throughout.

4) The qPCR primer design should be changed so that the primers span across exons to reduce the possibility of genomic signal contamination.

As instructed in the RNAqueous Micro Kit (Invitrogen AM1931), DNA is removed by enzymatic digestion. To ensure that the primer used was not detecting genomic signal contamination and was specific to cDNA, the qPCR was run using isolated RNA (with prior DNA removal or not) without cDNA synthesis, and this control had no amplification. Therefore, we conclude that the primer is specifically amplifying cDNA and does not amplify DNA or RNA prior to cDNA synthesis.

5) The species should be indicated in the title.

We revised the title to include the species.

https://doi.org/10.7554/eLife.42156.021

Article and author information

Author details

  1. Miae Jang

    The Department of Cellular and Integrative Physiology, University of Texas Health Science Center, San Antonio, United States
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Visualization, Writing—original draft
    Competing interests
    No competing interests declared
  2. Elizabeth Gould

    The Department of Cellular and Integrative Physiology, University of Texas Health Science Center, San Antonio, United States
    Contribution
    Data curation, Validation, Visualization
    Competing interests
    No competing interests declared
  3. Jie Xu

    1. The Department of Cellular and Integrative Physiology, University of Texas Health Science Center, San Antonio, United States
    2. Children's Medical Center, The Second Xiangya Hospital, Central South University, Changsha, China
    Contribution
    Data curation, Formal analysis, Validation, Investigation
    Competing interests
    No competing interests declared
  4. Eun Jung Kim

    The Department of Cellular and Integrative Physiology, University of Texas Health Science Center, San Antonio, United States
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Visualization
    Competing interests
    No competing interests declared
  5. Jun Hee Kim

    The Department of Cellular and Integrative Physiology, University of Texas Health Science Center, San Antonio, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    kimjh@uthscsa.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0207-8410

Funding

National Institute on Deafness and Other Communication Disorders (R01 DC03157)

  • Jun Hee Kim

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We would like to thank Drs. Klaus Nave and Manzoor Bhat for providing the Cnpcre 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.

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).

Senior Editor

  1. Gary L Westbrook, Vollum Institute, United States

Reviewing Editor

  1. Dwight E Bergles, Johns Hopkins University School of Medicine, United States

Reviewer

  1. Lu-Yang Wang, Hospital for Sick Children, Canada

Publication history

  1. Received: September 21, 2018
  2. Accepted: April 17, 2019
  3. Accepted Manuscript published: April 18, 2019 (version 1)
  4. Version of Record published: May 7, 2019 (version 2)

Copyright

© 2019, Jang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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