Slice preparation and electrophysiology

C57BL6 mice (Charles River Laboratories) from postnatal day 8 to 12, of either sex were used for all experiments described. The mice were housed in a facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care International, and protocols used for handling and care were reviewed by the Rutgers University Animal Care and Facilities Committee. Animals were decapitated without prior anesthesia, in accordance with NIH guidelines. Transverse brainstem slice thickness varied from 100 μm (immunohistochemistry and imaging) to 180 μm (electrophysiology) and were generated using a Leica VT1200 vibratome. Throughout the process of dissection and slicing, the brain was maintained in a low-calcium artificial CSF (aCSF) solution at 1–2°C containing the following (in mM): 125 NaCl, 25 NaHCO₃, 2.5 KCl, 1.25 NaH₂PO₄, 25 glucose, 0.8 ascorbic acid, three myo-inositol, 2 Na-pyruvate, 3MgCl₂, and 0.1 CaCl₂, pH 7.4, when oxygenated with carbogen gas (95% oxygen, 5% carbon dioxide). Once produced, slices were transferred to a holding chamber maintained at ~35°C for 30–40 min in normal calcium aCSF solution with the same composition listed above except for 1 mM MgCl₂ and 2 mM CaCl₂. This same solution was also used as the standard recording solution for electrophysiology experiments (see below). All experiments were performed at room temperature (22–25°C) for up to ~4–5 hr after the recovery period.

Patch-clamp recordings were conducted using an EPC10 USB double patch-clamp amplifier with PatchMaster software (HEKA; Harvard Bioscience). A transverse slice orientation was used in all postsynaptic voltage-clamp recordings in order to maintain the integrity of the calyceal axons for fiber stimulation. Calyx synapses in the medial nucleus of the trapezoid body (MNTB) were afferently stimulated (A-M Systems Isolated Pulse Stimulator Model 2100) using a bipolar fiber stimulator (lab design) placed at the midline of the slice. The MNTB field was scanned with an extracellular pipette to locate neurons that respond to midline fiber stimulation. For whole-cell recording, patch pipettes were produced from thick-walled borosilicate glass, 2.0 mm outer diameter, 1.16 mm inner diameter (Sutter Instruments). Postsynaptic pipettes (2–3 MΩ) were filled with a solution containing (in mM): 125 Cs-methanesulfonate, 20 CsCl, 20 TEA, 10 HEPES, five phosphocreatine (Alpha Aesar), 4 ATP, 0.3 GTP, and 2 QX-314 Cl^- (Sigma Aldrich; to block voltage gated Na⁺ channels on the postsynaptic neuron to measure the true EPSC) and was buffered to pH 7.4 using CsOH. To inhibit protein synthesis, anisomycin (Sigma, A9789) and emetine dihydrochloride (EMD Millipore, Calbiochem, 324693). Postsynaptic series resistances (R_s) for voltage clamp recordings was less than 15 MΩand typically varied less than 2 MΩ throughout the recording. For the recordings that appear in Figures 5, ​,66 and ​and7,7, showing EPSC response properties in control and after anisomycin treatment, the average R_s values and corresponding peak EPSC amplitudes in response to stimulation at 100 Hz and 200 Hz are graphed (see Figure 6—figure supplement 4A,B). We also provide a graph of Rs values and corresponding EPSC peak amplitude for the data in Figures 6 and ​and77 (see Figure 6—figure supplement 4C,D,E,F). In addition, an R_s compensation of 75–80% was applied for all recordings such that the adjusted R_s was in the range of 2–5 MΩ. Cells for which these criteria could not be applied, or maintained, were excluded from analysis. Recordings were acquired at sampling frequencies of 20 KHz and filtered by a 4-pole Bessel filter at 3 kHz. Holding potentials were set to −65 mV; junction potentials, calculated to be −11 mV, were not corrected.

For all electrophysiology recordings and data analysis measurements, anisomycin treatment and control conditions were blinded. In addition, the quality of the slices, neurons, and general recording conditions were determined by 1–2 initial recordings in normal aCSF. If the initial recordings had stable responses that lasted the duration of the stimulus protocols, a minimum of 25 min, then the recording solution was switched to a blinded cylinder of recording solution for subsequent recordings which were performed after ~1 hr of treatment (45 min to 120 min) in the absence of fiber stimulation. Since spontaneous action potentials are not present in these recording conditions, only spontaneous release activity occurred during the treatment period. On each day, the blinded cylinder would contain either: 40 μM anisomycin (Sigma Aldrich) or DMSO alone (vehicle). To test the effect of the translational inhibitor anisomycin (40 μM) on synaptic response characteristics, slices were preincubated for ~1 hr in the presence of the drug in the absence of afferent fiber stimulation. At all times, aCSF was continuously circulated using a peristaltic pump; total volume of the solution was 30 mL. All recordings, control and test conditions, were made in the presence of 25 μm bicuculline and 2 μm strychnine to block inhibitory responses. Power analysis to determine the appropriate sample size was performed based on means and standard deviation values of preliminary data. Recordings from 5 cells in each condition was estimated to be adequately powered, for α = 0.5, and a 0.8 power of test.

Miniature excitatory post-synaptic currents (mEPSCs) were recorded during 30 s continuous recordings at several times during the stimulation protocol. mEPSCs were analyzed by Mini Analysis Software (Synaptosoft, RRID:SCR_002184). The following mEPSC search parameters were used: gain, 20; blocks, 3940; threshold, 10 pA; period to search for a local maximum, 20,000 μsec; time before a peak for baseline, 5000 μsec; period to search a decay time, 5000; fraction of peak to find a decay time, 0.5; period to average a baseline, 2000 μsec; area threshold, 10; number of points to average for peak, 3; direction of peak, negative). Analysis was performed using the above settings, and visually checked to ensure accuracy. Evoked response traces were exported to Igor Pro (Wavemetrics, Portland, OR), and measurements were made manually, or using Taro Tools (Igor macro, Taro Ishikawa) with visual inspection and adjustment as necessary for every measured peak amplitude.

Data are presented as mean ± standard error of the mean (SEM). A Student’s t-test (MS Excel) was employed to determine if statistically significant differences exist between treated and control conditions. While the t-test is considered to be robust, data that do not have a normal distribution can affect the p-value. Therefore, an Anderson-Darling test (MATLAB) for normal distribution was run on each dataset used in the t-test comparisons. The null hypothesis for this test is that the dataset is normally distributed. Typically, α is set at 0.05, and we provide p-values for all Anderson-Darling test p-values < 0.1. Therefore, unless otherwise noted, p > 0.1 for each set of data. To further account for the possibility that the distribution of a dataset could affect the t-test value, the two-sample Kolmogorov-Smirnov (KS2) test (MATLAB), a nonparametric method, was also used to determine statistical significance. For the paired data comparisons, a two-sided Wilcoxon signed rank test (MATLAB) was used as a nonparametric test. For the repetitive stimulation data, a two way ANOVA (MS Excel) with repeated measures was used to compare the normal and treated responses. To reduce complications for running an unbalanced ANOVA, in a single data set (100 Hz anisomycin), the values from two recordings done on one day were averaged to allow an equal number of recordings to run a balanced two way ANOVA. Calculated p-values are indicated in relevant figures as follows: p ≤ 0.05 is considered significant (*); p ≤ 0.01 very significant (**); and p ≤ 0.001 highly significant (***). We define biological replicates as each tested cell (number of recordings), and technical replicates as multiple tests on a single cell. In our experiments, a minimum of four recordings, of spontaneous activity, 30 s each, were made during the recording time. Data were analyzed as initial spontaneous release levels, and spontaneous release following activity as described in the text. Outlier data for spontaneous event recordings resulted in removal of two cells from the data, as determined by Grubb’s test with α = 1%. The two-sample Kolmogorov-Smirnov (KS2) test was used to calculate the p-value for the cumulative probabilities of the mEPSC event intervals for two different conditions. Briefly, this nonparametric test uses the maximum vertical difference between two cumulative probability graphs and the total number of measurements to determine the statistical significance of the differences between two cumulative probability distributions. Histograms with identical bin-ranges were used to compare the mEPSC intervals for the two different conditions. This calculation was preformed manually, and by the KS function in MATLAB, which gave very similar or identical values.

Vesicle release properties were measured by plotting the cumulative response for each recorded response to 100 and 200 Hz stimulation. A best fit line through the cumulative EPSC responses to stimuli 15 to 30 (Figure 7A₁ and B₁) provides the slope of the steady state response corresponding to the vesicle replenishment rate; and the y-intercept of this line provides a measurement of the readily releasable pool (Schneggenburger et al., 1999; Neher, 2015). The initial probability of release was measured by dividing the peak amplitude of the first EPSC response by the corresponding RRP measured for that train (Schneggenburger et al., 1999; Neher, 2015). As an additional method to measure the initial probability of release and the readily releasable pool, the peak response was plotted against the stimulation number and a single exponential was fitted to the data (Thanawala and Regehr, 2016). The tau (τ) of the exponential fit was used to calculate the probability of release (Pr) using the formula: Pr = (1−e−1/τ). In instances where the response to the first stimulation (R₀) was smaller than the value predicted by the fitted exponential (RP₀), a facilitation correction (Fc) value was calculated by dividing the predicted value by the actual value: Fc = RP₀ / R₀, and Pr was divided by Fc to compensate for facilitation.