Inhibitory glycinergic neurotransmission in the mammalian auditory brainstem upon prolonged stimulation: short-term plasticity and synaptic reliability

Abstract

Short-term plasticity plays a key role in synaptic transmission and has been extensively investigated for excitatory synapses. Much less is known about inhibitory synapses. Here we analyze the performance of glycinergic connections between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) in the auditory brainstem, where high spike rates as well as fast and precise neurotransmission are hallmarks. Analysis was performed in acute mouse slices shortly after hearing onset (postnatal day (P)11) and 8 days later (P19). Stimulation was done at 37°C with 1-400 Hz for 40 s. Moreover, in a novel approach named marathon experiments, a very prolonged stimulation protocol was employed, comprising 10 trials of 1-min challenge and 1-min recovery periods at 50 and 1 Hz, respectively, thus lasting up to 20 min and amounting to >30,000 stimulus pulses. IPSC peak amplitudes displayed short-term depression (STD) and synaptic attenuation in a frequency-dependent manner. No facilitation was observed. STD in the MNTB-LSO connections was less pronounced than reported in the upstream calyx of Held-MNTB connections. At P11, the STD level and the failure rate were slightly lower within the ms-to-s range than at P19. During prolonged stimulation periods lasting 40 s, P19 connections sustained virtually failure-free transmission up to frequencies of 100 Hz, whereas P11 connections did so only up to 50 Hz. In marathon experiments, P11 synapses recuperated reproducibly from synaptic attenuation during all recovery periods, demonstrating a robust synaptic machinery at hearing onset. At 26°C, transmission was severely impaired and comprised abnormally high amplitudes after minutes of silence, indicative of imprecisely regulated vesicle pools. Our study takes a fresh look at synaptic plasticity and stability by extending conventional stimulus periods in the ms-to-s range to minutes. It also provides a framework for future analyses of synaptic plasticity.

Keywords: fast-spiking cells; high-frequency neurotransmission; inhibitory postsynaptic currents; lateral superior olive; medial nucleus of the trapezoid body; short-term depression; synaptic attenuation; synaptic fidelity.

Figure 1

Whole-cell patch clamp recordings from LSO neurons located in the central region of the nucleus upon electrical stimulation of their input fibers from the MNTB. (A) Schematic view of the circuit as seen in a coronal brainstem slice. A theta glass electrode was used for focal electrical stimulation and placed at the lateral border of the MNTB to reach the maximum number of connecting fibers (depicted in magenta). Glycinergic transmission was pharmacologically isolated with the depicted drugs. (B₁,B₂) Scheme of the two protocols used for prolonged and very prolonged stimulation, comprising up to 30,600 pulses.

Figure 2

Frequency-dependent changes in evoked glycinergic IPSCs obtained at P11 and physiological temperature (37 ± 1°C). (A₁) Original 40 s recording of a representative LSO neuron during 1 Hz stimulation (top). The first 550 ms are time-expanded in the bottom trace (see red-stippled line). The last IPSC during the 40-s-period is shown on the right and depicted by its pulse number (#40). The 200 pA calibration bar is also valid for panels (A₂,A₃). (A₂,A₃) IPSCs during 10 and 100 Hz stimulation, respectively, from the same neuron as in panel (A₁). Similar to (A₁), the very last IPSC during the 40-s-period is shown on the right (#400 and #4000, respectively). (B₁) Time course of IPSC peak amplitudes from three LSO neurons at eight stimulation frequencies, ranging from 1 to 333 Hz (for color code, see inset). Peak values to the first 40 pulses (#1–40) are plotted. (B₂) Like (B₁), but depicting the time course of IPSC peak amplitudes during the complete 40-s-periods. For each stimulation frequency, peak amplitudes were sampled at 1-s-intervals. Notice the frequency-dependent short-term depression (STD).

Figure 3

Population data illustrating the frequency-dependent time course of glycinergic IPSC peak amplitudes at P11/37°C. (A₁–A₈) Color-coded plots for all recorded LSO neurons at eight stimulation frequencies (n = 4–22, cf. inset in panel B₁), depicting the time course of IPSC amplitudes in response to the first 40 stimuli. In each panel, the mean values ± s.e.m. are shown in black. The failure rate (%) and the ratio failures/events across neurons are also provided in each panel. (B₁) Mean values ± s.e.m. of the first 40 IPSCs obtained in response to the eight different stimulation frequencies. Notice the decline to <40% at frequencies ≥100 Hz. (B₂) Mean values ± s.e.m. obtained during the complete 40-s-periods of stimulation (sampled at 1-s-intervals). Color code as in panel (B₁). Notice the decline to <20% at frequencies ≥100 Hz. Black frame depicts the last 10 data points in each trace that underwent statistical analysis shown in panel (C). (C) IPSC peak amplitudes decreased with increasing stimulation frequency. N numbers in the inset of (B₁) correspond to all panels. ^**p < 0.01; ^***p < 0.001.

Figure 4

Frequency-dependent changes in evoked glycinergic IPSCs obtained at P11 and room temperature (26 ± 2°C). (A₁) Original 40 s recording of a representative LSO neuron during 1 Hz stimulation (top). The first 550 ms are time-expanded in the bottom trace (see red-stippled line). The last IPSC during the 40-s-period is shown on the right and depicted by its pulse number (#40). The 200 pA calibration bar is also valid for panels (A₂,A₃). (A₂,A₃) IPSCs during 10 and 100 Hz stimulation, respectively, from the same neuron as in panel (A₁). Similar to (A₁), the last IPSC during the 40-s-period is shown on the right (#400 and #4000, respectively). (B₁) Time course of IPSC peak amplitudes from two LSO neurons at stimulation frequencies ranging from 1–200 Hz (for color code, see inset). Peak values to the first 40 pulses (pulse #1–40) are plotted. (B₂), Like (B₁), but depicting the time course of IPSC peak amplitudes during the complete 40-s-periods. For each stimulation frequency, peak amplitudes were sampled at 1-s-intervals. Notice the stronger amount of STD than at 37°C, particularly at higher frequencies.

Figure 5

Population data illustrating the frequency-dependent time course of glycinergic IPSC peak amplitudes at P11/26°C. (A₁–A₇) Color-coded plots for all recorded LSO neurons at seven stimulation frequencies (n = 4–18, cf. inset in panel B₁), depicting the time course of IPSC amplitudes during the first 40 stimulus pulses. In each panel, the mean values ± s.e.m. are shown in black. The failure rate (%) and the ratio failures/events across neurons are also provided in each panel. (B₁) Mean values ± s.e.m. of the first 40 IPSCs obtained in response to different stimulation frequencies. Notice the decline to <40% at frequencies ≥100 Hz. (B₂) Mean values ± s.e.m. obtained during the complete 40-s-periods of stimulation (sampled at 1-s-intervals). Color code as in panel B₁. Notice the virtual collapse to <10% at frequencies ≥50 Hz. Black frame depicts the last 10 data points in each trace that underwent statistical analysis shown in panel (C). (C) IPSC peak amplitudes decreased with increasing stimulation frequency more profoundly than at P11/37°C. N numbers in the inset of (B₁) correspond to all panels. ^**p < 0.01; ^***p < 0.001.

Figure 6

Frequency-dependent changes in evoked glycinergic IPSCs obtained at P19/37°C. (A₁) Original 40 s recording of a representative LSO neuron during 1 Hz stimulation (top). The first 550 ms are time-expanded in the bottom trace (see red-stippled line). The last IPSC during the 40-s-period is shown on the right and depicted by its pulse number (#40). The 200 pA calibration bar is also valid for panels (A₂,A₃). (A₂,A₃), IPSCs during 10 and 100 Hz stimulation, respectively, from the same neuron as in panel (A₁). Similar to (A₁), the last IPSC during the 40-s-period is shown on the right (#400 and #4000, respectively). (B₁) Time course of IPSC peak amplitudes from two LSO neurons at eight stimulation frequencies, ranging from 1–400 Hz (for color code, see inset). Peak values to the first 40 pulses (pulse #1–40) are plotted. (B₂) Like (B₁), but depicting the time course of IPSC peak amplitudes during the complete 40-s-periods. For each stimulation frequency, peak amplitudes were sampled at 1-s-intervals.

Figure 7

Population data illustrating the frequency-dependent time course of glycinergic IPSC peak amplitudes at P19/37°C. (A₁–A₉), Color-coded plots for all recorded LSO neurons at nine stimulation frequencies (n = 7–15, cf. inset in panel B₁), depicting the time course of IPSC amplitudes during the first 40 stimulus pulses. In each panel, the mean values ± s.e.m. are shown in black. The failure rate (%) and the ratio failures/events across neurons are also provided in each panel. (B₁) Mean values ± s.e.m. of the first 40 IPSCs obtained in response to different stimulation frequencies. Notice the decline to <50% at frequencies ≥50 Hz. (B₂) Mean values ± s.e.m. obtained during the complete 40-s-periods of stimulation (sampled at 1-s-intervals). Color code as in panel (B₁). Notice the gradual decline with frequency up to 200 Hz. Black frame depicts the last 10 data points in each trace that underwent statistical analysis shown in panel (C). (C) IPSC peak amplitudes decreased with increasing stimulation frequency, yet stayed at >30% of the control amplitude up to 100 Hz. N numbers in the inset of (B₁) correspond to all panels. ^**p < 0.01; ^***p < 0.001.

Figure 8

Temperature dependency and age dependency of IPSC kinetics. (A) Average of 40 IPSCs obtained by 1 Hz stimulation at P11/26°C (black), P11/37°C (red), and P19/37°C (blue). Each trace is obtained from a single, representative MNTB-LSO connection. 0 ms corresponds to stimulus onset. Inset depicts traces with aligned peak amplitudes to better visualize differences in rise and decay time. (B) Statistical analysis of rise time, decay time, and peak amplitude. Numbers in bars depict the number of analyzed neurons, circles in bars depict single values. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 9

Temperature dependency of STD and recovery of IPSC peak amplitudes at P11. Neurons were stimulated with 50 Hz for 60 s (depression phase) and subsequently stimulated with 1 Hz for another 60 s (recovery phase). (A₁,B₁) Time course of the IPSC peak amplitudes of two representative LSO neurons at 26°C (A₁) and 37°C (B₁). Insets show eight original IPSCs at the positions marked by encircled numbers. (A₂,B₂), Time course of the IPSC peak amplitudes of all recorded neurons. Mean values ± s.e.m. are shown in black (A₂: 26°C, n = 17) and red (B₂: 37°C, n = 22). The mean values represent the simple moving average of five data points, thus smoothing out short-term fluctuations and highlighting longer-term trends. (C) Superposition of average time courses to highlight the temperature effects. Mono-exponential fits during the recovery phase were obtained for each condition (green). Data obtained during seconds 50–60 and 110–120 (cf. bars) were used for the statistical analysis shown in panel (D). (D) At 26°C, peak amplitudes declined significantly stronger during the depression phase. The opposite finding was obtained for the recovery phase. (E) The recovery was significantly faster at 37°C. τ-values were obtained by fitting the time course for each individual neuron. Numbers in bars depict numbers of neurons analyzed. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 10

Time course of depression and recovery of IPSC peak amplitudes at P11/37°C upon very prolonged stimulation, lasting 20 min (cf. Figure 1B₂). Ten trials of 50 and 1 Hz/60 s episodes were applied (Trial 1–10), amounting to a total of 30,600 stimuli. (A) Time course of the IPSC peak amplitudes (one color per neuron, n = 21–22). Mean values ± s.e.m. are shown in black (simple moving average of five data points). Due to the smoothing, failures are no longer discernable. (B) Superposition of the ten averaged time courses, illustrating the similarities across trials (traces for trials 1, 5, and 10 are highlighted by a thick black, turquoise, and magenta line, respectively). Statistical comparison was done between trials 1 and 5 and trails 1 and 10. ^*p < 0.05.

Figure 11

Time course of depression and recovery of IPSC peak amplitudes at P11/26°C upon very prolonged stimulation. Same procedure as in Figure 10, except that recordings were obtained at 26°C. (A) Time course of the IPSC peak amplitudes (one color per neuron, n = 17). Mean values ± s.e.m. are shown in black (simple moving average of five data points). (B) Superposition of the ten averaged time courses, illustrating the differences between trials, particularly during the recovery phases (traces for trials 1, 5, and 10 are highlighted by a thick black, turquoise, and magenta line, respectively). Statistical comparison was done between trials 1 and 5 and trails 1 and 10. ^**p < 0.01; ^***p < 0.001.

Figure 12

Temperature effect on synaptic attenuation and recovery as assessed during very prolonged stimulation (20 min). (A) Superposition of averaged time courses depicted in Figures 10, 12 (26°C in black, 37°C in red), highlighting the temperature-dependent differences. (B) Statistical analysis of IPSC peak amplitudes at the end of the challenge periods (50–60 s). (C) As in (B), but for the end of the recovery periods (110–120 s). (D) Failure rate analysis at the beginning (0–800 ms) and end (59–60 s) of the challenge periods. (E) As in (D), but at the beginning (60–70 s) and end (110–120 s) of the recovery periods. ^**p < 0.01; ^***p < 0.001.

Figure 13

Fidelity analysis of action potentials evoked through orthodromic (A–C) and antidromic (D–F) stimulation of P11/37°C MNTB axons. Orthodromic stimulation was performed as in Figure 1B₁, and recordings were obtained from LSO neurons. (A₁,A₂) Original current traces from a neuron stimulated with 100 Hz, depicting IPSCs to stimulus number 31–40 (A₁) and to stimulus number 3991–4000 (A₂). Dots mark reliable synaptic transmission; note six failures in (A₂). (B₁) Dot plots from the LSO neuron in (A) stimulated with seven frequencies (1–200 Hz), illustrating fidelity behavior to stimulus number 1–40. Frame at 100 Hz depicts the scenario shown in (A₁). Numbers to the right are the numbers of successful responses. (B₂) Dot plots from the same LSO neuron, again stimulated at seven frequencies (1–200 Hz), but now illustrating fidelity behavior to stimulus number 3961–4000. Frame at 100 Hz depicts the scenario shown in (A₂). Notice the occurrence of failures at frequencies ≥50 Hz. (C₁,C₂), Fidelity data of the population of LSO neurons as a function of stimulus frequency. 100% fidelity means 40 successful responses to 40 stimuli (40/40). Individual neurons are depicted in different colors, and mean values ± s.e.m. are shown in black. For antidromic stimulation, MNTB axons were stimulated in the LSO and somatic recordings were obtained from MNTB neurons. (D₁,D₂) Original voltage traces from a neuron stimulated with 100 Hz, depicting action potentials to stimulus number 11–20 (D₁) and to stimulus number 3971–3980 (D₂). Dots mark successful antidromic propagation; note five failures in (D₂). (E₁) Dot plots from the MNTB neuron in (D) stimulated with seven frequencies (1–200 Hz), illustrating fidelity behavior to stimulus number 1–40. Frame at 100 Hz depicts the scenario shown in (D₁). Numbers to the right are the numbers of successful spike propagation. (E₂) Dot plots from the same MNTB neuron, again stimulated at seven frequencies (1–200 Hz), but now illustrating fidelity behavior to stimulus number 3961–4000. Frame at 100 Hz depicts the scenario shown in (D₂). Notice the occurrence of an increasing number of failures at frequencies ≥100 Hz. (F₁,F₂) Fidelity data of the population of LSO neurons (n = 7) as a function of stimulus frequency. 100% fidelity means 40 successful responses to 40 stimuli (40/40). Mean values ± s.e.m. are shown by black squares. Notice that the course is similar to that seen upon orthodromic stimulation.