Specific synaptic input strengths determine the computational properties of excitation-inhibition integration in a sound localization circuit

Abstract

Key points: During the computation of sound localization, neurons of the lateral superior olive (LSO) integrate synaptic excitation arising from the ipsilateral ear with inhibition from the contralateral ear. We characterized the functional connectivity of the inhibitory and excitatory inputs onto LSO neurons in terms of unitary synaptic strength and convergence. Unitary IPSCs can generate large conductances, although their strength varies over a 10-fold range in a given recording. By contrast, excitatory inputs are relatively weak. The conductance associated with IPSPs needs to be at least 2-fold stronger than the excitatory one to guarantee effective inhibition of action potential (AP) firing. Computational modelling showed that strong unitary inhibition ensures an appropriate slope and midpoint of the tuning curve of LSO neurons. Conversely, weak but numerous excitatory inputs filter out spontaneous AP firing from upstream auditory neurons.

Abstract: The lateral superior olive (LSO) is a binaural nucleus in the auditory brainstem in which excitation from the ipsilateral ear is integrated with inhibition from the contralateral ear. It is unknown whether the strength of the unitary inhibitory and excitatory inputs is adapted to allow for optimal tuning curves of LSO neuron action potential (AP) firing. Using electrical and optogenetic stimulation of afferent synapses, we found that the strength of unitary inhibitory inputs to a given LSO neuron can vary over a ∼10-fold range, follows a roughly log-normal distribution, and, on average, causes a large conductance (9 nS). Conversely, unitary excitatory inputs, stimulated optogenetically under the bushy-cell specific promoter Math5, were numerous, and each caused a small conductance change (0.7 nS). Approximately five to seven bushy cell inputs had to be active simultaneously to bring an LSO neuron to fire. In double stimulation experiments, the effective inhibition window caused by IPSPs was short (1-3 ms) and its length depended on the inhibitory conductance; an ∼2-fold stronger inhibition than excitation was needed to suppress AP firing. Computational modelling suggests that few, but strong, unitary IPSPs create a tuning curve of LSO neuron firing with an appropriate slope and midpoint. Furthermore, weak but numerous excitatory inputs reduce the spontaneous AP firing that LSO neurons would otherwise inherit from their upstream auditory neurons. Thus, the specific connectivity and strength of unitary excitatory and inhibitory inputs to LSO neurons is optimized for the computations performed by these binaural neurons.

Keywords: auditory system; excitation-inhibition integration; glutamate; glycine; optogenetics; synaptic transmission.

Figure 1

LSO neurons receive large inhibitory inputs Aa, IPSCs recorded with a high [Cl⁻]_i pipette solution, in response to increasing intensities of electrical stimulation. Holding potential was −70 mV. Ab, peak IPSC amplitudes plotted as a function of stimulus intensity. Horizontal lines indicate the average amplitude of each identified step. The arrow indicates a fluctuation of IPSC amplitudes evoked under a constant stimulation intensity, indicating that this stimulus intensity was at the threshold for recruiting an additional fibre (N = 8 repetitions at 0.1 Hz for each stimulation intensity). Ac, example traces from the recording in (Aa) before (in grey) and after offline R _s compensation (black or red trace). For the larger IPSC, offline R _s compensation resulted in a larger than 50% amplitude increase (red trace, and red data points in Ab). The data in (Aa) to (Ac) are from the same recording. Ba and Bb, example IPSC input–output curve from another LSO neuron. In this example, a more gradual IPSC increase was apparent at higher stimulation intensities. C, histograms of unitary IPSC amplitudes for each recorded cell. Note that, for cells #3 and #7, steps could not be identified for all parts of the input–output curve, and the more graded IPSC increase is reported as a dotted line. The example recordings shown in (A) and (B) correspond to cells #9 and #7, respectively. D, histogram of the unitary IPSC amplitudes combined from all recordings (mean ± SD: 2.37 ± 2.1 nA; n = 42 unitary IPSCs from n = 9 cells; bin size = 200 pA). Note that the distribution is right‐skewed. E, histogram of all the unitary IPSC amplitudes, logarithmized, and fitted with a Gaussian function. F, normal probability plot of the logarithmized unitary IPSC amplitudes. Note that the majority of the data points lie close to the unitary line (except for the smallest and largest values), consistent with a normal distribution. G, plot of the unitary IPSC amplitudes in their order of appearance, from lowest to highest stimulation intensity, for n = 9 recordings. Average values for the first (left) and the last (right) unitary IPSC are also shown (grey mean ± SD data points).

Figure 2

Inhibitory conductances at a physiological intracellular Cl⁻ concentration Aa, IPSCs recorded with low [Cl⁻]_i (6 mM), evoked by increasing electrical stimulation intensities as indicated. Holding potential was −50 mV. Ab, input–output curve for the IPSCs recorded in (Aa). Ba, IPSCs recorded under low [Cl⁻]_i, at holding potentials ranging of −90 mV, −85 mV and −80 mV and then increments of + 10 mV. Each trace is the average of n = 8 IPSCs. Bb, I–V plots of IPSCs recorded under low [Cl⁻]_i conditions (left data points, same recording as in A and Ba), and under high [Cl⁻]_i from a different cell. C, reversal potentials obtained for the IPSCs measured under high (n = 2 cells) and low [Cl⁻]_i solutions (n = 5 cells). Horizontal bars indicate the average. D, average and individual values of the conductance of the unitary IPSCs for the data measured under low [Cl⁻]_i (left). The data on the right represent the conductance values of unitary IPSCs under high [Cl⁻]_i, converted from the measurements of unitary IPSC shown in Fig. 1. Note the significantly smaller unitary IPSCs under conditions of low [Cl⁻]_i (P < 0.0037; Mann–Whitney test).

Figure 3

Minimal optogenetic stimulation of PV‐positive fibres reveals large unitary IPSCs A, scheme of the experimental design, in which blue light pulses were delivered onto the LSO (or sometimes MNTB) in PV^Cre x ChR2 mice. B, immunohistochemistry performed in a PV^Cre x ChR2 mouse at P21 at the level of the LSO, with antibodies against VGAT (red channel) and GFP to localize ChR2‐eYFP (green channel). Scale bar = 5 μm. Ca and Cb, light‐evoked IPSCs obtained with 1 ms light pulses onto LSO (blue trace) at increasing light intensities. In (Cb), the holding potential (V_hold) was lowered to −20 mV. D, plot of the latency of the optogenetically evoked IPSCs as a function of LED light intensity, fitted with a single‐exponential function. Data points are from the recording in (C). Ea and Eb, minimal optogenetic stimulation in the LSO with either 1 ms long pulses (left and middle panel) or with prolonged (5 ms) but even dimmer light pulses (right). IPSCs are shown with a continuous line before and with a dotted line after a second peak occurs. F, histogram of the amplitudes of the first IPSC peaks in (E), fitted with a multipeak Gaussian function (n = 83 events). G, plot of the unitary IPSC amplitudes for each recorded cell, estimated by optogenetic stimulation in the LSO. For the first five cells, a subsequent optogenetic stimulation was performed in the MNTB (see K). Ha, histogram of the unitary IPSC amplitudes combined from all recordings. Hb, histogram of the logarithmized unitary IPSC amplitudes. The distribution can be fitted with a Gaussian curve, except for the very small values. Hc, normal probability plot of the logarithmized unitary IPSC amplitudes. Note that the majority of the data points lie close to the unitary line, consistent with a log‐normal distribution. I, IPSCs recorded in the same neuron as shown in (C) and (E), but with the optical stimulation moved to the MNTB area, for two different light intensities (left, and right). J, histogram of all the first peak IPSC amplitudes with light stimulation in the MNTB, fitted with a multipeak Gaussian function (n = 104 events). K, plot of the unitary IPSC amplitudes for each recorded cell, estimated by optogenetic stimulation in the MNTB. L, average values of the unitary IPSC amplitudes evoked with light stimulation in the LSO (black) or MNTB (red; n = 45 and 29, respectively). Note the significantly smaller unitary IPSCs evoked by optogenetic stimulation in the MNTB (P < 0.0005; Mann–Whitney test). Error bars indicate the SD.

Figure 4

Optogenetic stimulation of the excitatory synapses under the PV‐promoter reveals small unitary EPSC amplitudes Aa, light‐evoked EPSCs obtained with 1 ms light pulses to the surrounding LSO tissue at increasing intensities in a PV^Cre x ChR2 mouse. The recordings were performed in the presence of strychnine (2 μm) and bicuculline (10 μm) to block inhibitory inputs. The pink trace in the far left graph indicates a failure. The other traces obtained with the same light stimulation were offset for clarity. Note the early‐rising, but more slowly decaying light‐evoked photocurrent. Faster‐rising and decaying glutamatergic EPSCs are also observed. Ab, input–output curve for the same experiment. Note the discrete, but small first ‘step’ with weak light intensities (see inset). B, histogram of the EPSC amplitude measured at the threshold for successful stimulation in the same recording as shown in (Aa) and (Ab). The average of this distribution (30 ± 15 pA, n = 67 events) was taken as the estimate of the unitary EPSC. C, individual and average data points of unitary EPSC amplitudes. Error bars indicate the SD.

Figure 5

Optogenetic stimulation under the Math5 promoter reveals high convergence of bushy cell inputs to LSO neurons A, scheme of the experimental design, in which blue light stimulation was delivered to LSO tissue of Math^Cre x ChR2 mice. B, immunohistochemistry at the level of the LSO with an anti‐GFP antibody (green channel, to detect the transgenic ChR2‐eYFP), and an anti‐VGluT2 antibody (red channel, to detect glutamatergic nerve terminals). Scale bar = 5 μm. C, light‐evoked EPSPs (top traces) and EPSCs (bottom traces) recorded in the same example cell; light stimuli of increasing intensity were applied to the surrounding LSO tissue. Da, occurrence of APs and EPSP amplitudes plotted as a function of blue light intensity. A stimulus at a given intensity was repeated three times at 0.1 Hz. Db, EPSC amplitudes plotted as a function of stimulus light intensity. The data in (C) to (D) are from the same recording. E, input–output curves of optically evoked EPSCs for n = 7 cells. Each data point represents the average of n = 3 repeated stimuli at a given light intensity. F, average and individual data points of the unitary EPSC amplitude (left), and of the EPSC amplitude measured at the threshold of a reliably triggered AP (right). G, average and individual data points of the estimated number of inputs (left), and of the maximal EPSC amplitude (right).

Figure 6

The effective window for inhibition is brief and determined by the ratio of excitatory and inhibitory conductances Aa, top: response of an LSO neuron to optogenetic stimulation of bushy cell inputs in a Math5^Cre x ChR2 mouse at 10% of maximal LED intensity. Note the reliably evoked APs (curtailed at −20 mV). Bottom: response of the same LSO neuron to electrical stimulation of MNTB fibres (at 18 V), which generates an IPSP of around −15 mV, and an IPSC of 0.87 nA (red trace). Ab, IPSCs recorded in the same LSO neuron evoked by the two different stimulation intensities (5 and 18 V) used for the E–I integration experiment. B, E–I integration experiment: the timing of optogenetic stimulation (EPSP onset is indicated by a grey arrow) was varied relative to inhibition (indicated with a dashed line) to identify the optimal time difference for effective inhibition. The optogenetic stimulation of bushy cell fibres is indicated by blue bars (1 ms light pulses at 10%). The time differences between the onset of the IPSP and the onset of the EPSP are indicated (‘Δt’ values). Note that AP firing was suppressed (star symbols) when IPSP and EPSP occurred nearly synchronously. C, probability of AP generation plotted as a function of the time difference between the IPSP and EPSP onset. Red data points were obtained with the higher electrical stimulation intensity (18 V; IPSC of 0.87 nA or 33.5 nS), whereas the pink data points were obtained with the lower stimulation intensity (5 V; IPSC of 0.31 nA or 12.3 nS). Each data point was estimated from n = 5 repetitions. The width of the inhibition period is referred to as the effective inhibition window. The data in (A) to (C) were from the same recording. D, plot of the effective inhibition window as a function of the inhibitory conductance, for all cells investigated (n = 6). Data points linked by a continuous line represent recordings under the first (usually weaker) optogenetic stimulation of excitatory inputs, whereas data points linked by a dotted line represent recordings with the second (usually stronger) excitatory inputs. The resulting values for excitation strength, g _exc (nS), are indicated in the label (value for the second excitation strength, in brackets). E, same data as in (D) but plotted as a function of the ratio between the inhibitory and the excitatory conductance in each cell. The grey area highlights an observed effective inhibition window of 1–3 ms, for which ratios of inhibition over excitation of at least 2 were necessary. F, plot of the IPSC and IPSP half‐widths (empty and filled squares, respectively) and of the effective inhibition window (open circles), as a function of G _inh.

Figure 7

The strengths and numbers of unitary inhibitory and excitatory inputs are key determinants of the tuning curve and the spontaneous firing properties of LSO neurons A, schematic representation of the connectivity of excitatory and inhibitory inputs onto an LSO neuron as modelled in the present study. B, summary of the parameters used for the model simulations (for further details, see Methods). The parameters shown in bold are those of ‘case 1’ (D–G) and these connectivity‐related parameters were varied in the simulations; all other parameters were fixed. Ca, simulated firing rates of the excitatory and inhibitory afferents onto an LSO neuron (black and red traces, respectively), which function as input to the model. Cb, plot of the distribution of conductance values of n = 8 inhibitory inputs used for case 1 and 4. Da, simulated postsynaptic conductances in response to modelled input firing rates of N = 8 inhibitory and N = 40 excitatory inputs (red and black traces, respectively). Db, resulting membrane potential (V_m) trace (top), spike raster plot for 1000 repetitions (middle) and peri‐stimulus spike time histograms (bottom). Note the large hyperpolarizing fluctuations in the V _m trace caused by the strong unitary strength of inhibition. Inset: distribution of the V _m values under background input activity. Ea and Eb, similar display of simulation results as in (Da) and (Db), but now modelled for N = 40 inhibitory afferents, with proportionally reduced unitary IPSC strength (1.8 nS; ‘case 2’). Note the smaller fluctuations of V _m values (inset), and the absence of spiking responses with the 60 Hz/60 Hz stimulation, compared to case 1, and the smaller spontaneous firing visible in the spike raster plots in between stimulations. F, average spontaneous firing rates of the LSO neuron for the four modelled cases. G, evoked firing rates of the LSO neuron for three modelled cases, in response to excitatory inputs firing at 60 Hz and inhibitory inputs firing from 10 to 120 Hz, thus simulating varying sound intensity at the contralateral ear. H, spontaneous firing rates (left) and tuning curve (right) when the number of inhibitory inputs is varied over a larger range. I, spontaneous firing rates (left) and tuning curve (right) when the number of excitatory inputs is varied over a larger range. Note the strong influence of the excitatory convergence on spontaneous firing rates. J, evoked firing rates of the LSO neuron for the modelled cases 1 and 4. The error bars in (F) to (I) correspond to the SD.