To understand how auditory information is transmitted from the auditory nerve to the cochlear nuclei, we measured the short-term synaptic plasticity at the auditory nerve to nucleus angularis (NA) synapse. Whole cell patch-clamp recordings were made from NA neurons in chick embryonic (E16–E19) auditory brain stem slices in vitro, as described previously (MacLeod and Carr 2005
). By this age, synapse development is largely complete, and hearing has begun in ovo
(Kubke and Carr 2000
; Rubel and Parks 1988
). Synaptic responses were evoked by electrical stimulation of the 8th
(auditory) nerve with a metal bipolar electrode positioned medial to the nucleus, where nerve fibers enter the neuropil. We measured pharmacologically isolated AMPA-receptor mediated excitatory postsynaptic currents (EPSCs) under voltage-clamp in response to trains of stimuli. Stimulation consisted of 8–10 pulses in trains of a constant interpulse interval, and a recovery pulse delivered 2 s after the last pulse in the train. Six different interpulse intervals were used, corresponding to train frequencies of 10, 33, 100, 143, 200, and 250 Hz, which we refer to as input rate
to avoid confusion with acoustic frequency. These input rates were chosen to span the range of firing rates observed in the auditory nerve of young hatchling and embryonic chicks, which have spontaneous rates of approximately 0–80 Hz and maximal average driven rates of approximately 250–300 Hz (see the example in ; also: Jones and Jones 2000
; Salvi et al. 1992
; Saunders et al. 2002
Short-term plasticity of auditory nerve synapses in NA is composed of a mixture of depression and facilitation
In contrast to the profound short-term depression uniformly expressed at synapses in the brain stem pathways that encode timing information, we found that the short-term synaptic plasticity recorded at the auditory nerve to NA neuron synapses was typically composed of a mixture of facilitation and depression (). In this paper, we use the term “enhancement” to describe the measured net increase in the EPSC amplitude and the term “facilitation” to refer to the putative underlying synaptic molecular mechanism, presumed to be a presynaptic increase in release probability. Enhancement of the EPSC amplitude (above a criterion of 10% greater than initial EPSC amplitude) was observed in a subset of NA recordings for at least one stimulus rate (n = 12/34; ). For example, enhancement of the EPSCs recorded from one NA neuron during stimulus input trains of 100, 143, and 200 Hz is shown in . A maximal EPSC amplitude occurred on the 2nd or 3rd stimulus and some enhancement was sustained throughout the rest of the train (). For the 250 Hz input train, the EPSC amplitude declined after an initial enhancement back to the amplitude of the initial EPSC. In contrast, the EPSCs recorded during 10 Hz and 33 Hz input trains did not show any transient enhancement (), but instead depressed in amplitude to a steady state ratio of 0.6 and 0.8, respectively (, markers). In other recordings, the enhancement of EPSC responses was moderate, limited to less than a two-fold increase over the initial EPSC amplitude. The enhancement was often followed by a depression in the recovery EPSC in response to a stimulation delivered after a delay period (2 s after the end of the train; “R”, in ). This “anomalous” recovery response suggested that the EPSCs were the result of two competing mechanisms, one which facilitated the synaptic response and the other which depressed the synaptic response. In the case of enhancement during the train, the facilitating factor dominated. At the time of the recovery response, the facilitating factor had declined, and the depressing factor dominated. These effects could be reproduced with a quantitative model of synaptic plasticity, described below.
FIG. 1 Excitatory postsynaptic currents (EPSCs) recorded in 1 nucleus angularis neuron in the chick auditory brain stem during stimulation of the auditory nerve fiber inputs. A: average traces in response to stimulus trains of 8 pulses at 6 different input stimulus (more ...)
FIG. 2 A: mean EPSC amplitudes, normalized to the initial EPSC plotted vs. stimulus pulse number in the train (1–8; R, EPSC response after a 2-s recovery period). Same data as in . B: steady-state amplitude ratio plotted vs. stimulus input train (more ...)
The relationship of the EPSC amplitude with train input rate was frequently nonmonotonic
To more easily compare the short-term plasticity across different synapses, we characterized the synaptic transfer function, defined as the relationship between the steady state amplitude and the input stimulus rate. A majority of synapses in NA (n = 18/34) had synaptic transfer functions that were nonmonotonic: they generally had a peak steady state amplitude for 33 or 100 Hz input trains. The example in shows this nonmonotonic profile, a result of net enhancement of the EPSC during the train. Another example in , however, shows a synapse that also had a nonmonotonic profile, but in the absence of any net enhancement of the EPSC amplitude (). In this case, a relative enhancement at the 33 Hz input rate also resulted in a nonmonotonic profile (). To distinguish between the presence of an underlying facilitation that counterbalances the depression, or the simple lack of depression, we also measured the EPSC after a recovery period as in . In , the amplitude of the recovery EPSC was reduced relative to the EPSCs at the end of the 33 Hz train, suggesting that during the train a similar competition between depression and facilitation was occurring as in the enhancement case, but with a balance in favor of net depression.
FIG. 3 Recording in another NA neuron the synaptic inputs of which showed no transient or sustained facilitation. A: normalized EPSC amplitudes showed only net depression across all input rates. B: steady-state ratio profile showed a relative enhancement for (more ...)
For many synapses, the overall effect of these competing components was to maintain the unitary synaptic conductance across a broad range of input rates with little net enhancement or depression. For a minority of synapses, however, the synaptic transfer functions were similar to those found in the timing pathway: monotonically increased depression with increased input rate (n = 9/34; ). The monotonically depressing synapses also showed an “anomalous” recovery, that is, the higher input rate trains appeared to recover more quickly than the low rate trains (note the crossover in the recovery amplitudes in , in which the 200 Hz recovery EPSC amplitude exceeded that of the 10 Hz recovery EPSC). The classification of the remaining synapses (n = 8/34) was uncertain due to large variation in the responses or irregularity of the synaptic transfer function, but two had nonmonotonic, “bowl-shaped” transfer functions in which the intermediate input rates resulted in the greatest net depression. All synaptic transfer functions (n = 34) are shown in .
FIG. 4 Recording from a 3rd NA neuron the synaptic inputs of which showed only simple depression. A: normalized EPSC amplitudes showed only net depression across all input rates. B: steady-state ratio profile showed monotonically increasing depression with stimulus (more ...)
FIG. 6 Summary of response profiles vs. stimulus input rate in NA. A: paired-pulse ratio, using the 2nd EPSC in the train. B: steady-state amplitude ratio as in –. Thin gray lines represent response profiles from individual NA neurons; thick (more ...)
Data were well fit by a model of short-term plasticity
To determine whether the synaptic responses observed could be accounted for by a simple model of short-term plasticity, we fit each synapse individually with a dynamic model of synaptic release, similar to a class of models of plasticity published previously by a number of investigators (see METHODS; Abbott et al. 1997
; Dittman et al. 2000
; MacLeod and Horiuchi 2006
; Tsodyks and Markram 1997
). EPSCs were presumed to result from the fractional release of vesicles from a release-ready pool at the time of each presynaptic stimulation. The model contained both depression and facilitation. Depression resulted from a decrease in the size of the release-ready pool due to depletion, and recovery from depletion followed a two-step vesicle pool replenishment process. Facilitation was implemented by making the vesicle release fraction activity- and time-dependent. EPSCs were generated by the model for each stimulus in the train, and compared with the data for each individual neuron (; see also Supplemental Figures S1 and S21
). Using a randomly seeded gradient descent method to find the parameters, we were able to fit nearly all the NA cells (30 of 34; 4 were excluded due to data irregularity). The time-dependent variables in the model during the input trains are shown for fits to three NA synapses (panels C
in –; specific model parameters are given in the legend). In general, the data were well fit with a fast recovery time constant for the readily releasable pool (τk1
= 20.1 ± 16.0 ms, n
= 30), and a slow recovery time constant for the backup pool (τk2
= 8.4 ± 9.3 s, n
= 30; excluding one outlier at 80.9 s) (; ). Many NA profiles were best fit with some degree of facilitation (average τF
= 42.4 ± 30.0 ms, n
= 20; excluding one outlier of 542 ms). Other NA profiles were best fit by minimizing the facilitation, including all those that were classified as monotonically depressing (the Xs in clustered on far left). The initial release fraction (F0
, equivalent to an average probability of release) ranged from 0.07 to 0.92 (0.43 ± 0.21; n
= 30). The fit was considered good if the error was low (compared with a zero-plasticity baseline error), and if several other qualitative criteria were met. Besides a good overall fit to the train data, model should fit the recovery response, especially the “anomalous” recovery. For example, the recovery responses in show that long after the facilitation parameter had relaxed to its initial value (top dashed line), the release-ready vesicle pool had not yet fully recovered (solid line). This resulted in a model recovery EPSC that was smaller than the EPSCs during the train (black dot late in the trace), matching the recovery EPSCs in the data. Finally, the model synapses should reproduce the profile of the steady state function (solid lines in panel B
, –). Reduced models in which a single pool was modeled, the relative pool sizes were held to be equal, or facilitation was eliminated (for those that required it) produced poorer fits, or failed to meet the criteria listed above. Thus we were able to account for the wide range of synaptic plasticity profiles in our data sample with a simple quantitative model of vesicle release, and a restricted parameter range ().
Model fits of the NA synapses
FIG. 5 Parameters of the model fit of all NA neurons. A: depression parameters F0, the initial fraction of release, vs. τk1, the time constant of recovery of the readily releasable vesicle pool. B: facilitation parameters, δF, the fractional (more ...)
Population and average profiles
Summary data for all the train data obtained from NA recordings are shown in . Because there were no differences observed related to the cell types within NA (defined by their firing responses to current injection; see supplementary Fig. S3), the data are pooled here. The paired pulse ratios (i.e., the ratio of the 2nd EPSC in the train to the 1st EPSC) plotted in demonstrated that for most NA neurons, there was either enhancement (in 11/34 neurons) or generally weak paired pulse depression, in contrast to the strong paired pulse depression seen in NM and NL neurons (see following text, ). The average paired pulse ratio remained about 0.80–0.90 across all rates, with a slight maximum at 33–100 Hz (). The average steady state ratio profile with input rate was also nonmonotonic, similar to many individual steady state profiles, with a maximum at 33 Hz (0.77 ± 0.23, mean ± SD; ). The average steady state ratio decreased to 0.70 ± 0.18 at 10 Hz, and was minimal at 250 Hz at 0.52 ± 0.26.
FIG. 7 EPSCs recorded in nucleus magnocellularis (NM, A) and nucleus laminaris (NL, B) in response to trains of stimuli showed simple, strong depression and a monotonic relationship to input rate (see also ). Synaptic responses in laminaris were evoked (more ...)
Relationship with initial EPSC size
The average initial EPSC amplitude recorded in the NA neurons was 232.3 ± 212.4 pA, and ranged from 30.7 pA to 1113.3 pA (n
= 34). There was no correlation between the amplitude of the initial EPSC with the paired pulse or steady state ratio at any frequency. Thus smaller EPSC recordings were no more or less likely to demonstrate depression, or facilitation, than those of larger EPSCs. The size of the EPSC reflects of the number of inputs recruited with a given stimulus intensity, and is unrelated to release probability (MacLeod and Carr 2005
External calcium levels alters the paired pulse ratio
Although the paired pulse ratio was not related to initial EPSC amplitude, the ratio could be altered by changes in the external calcium concentration. We investigated the calcium dependence of the paired pulse ratio with a 10 ms interval. Application of an elevated calcium ACSF (4 mM) shifted the paired pulse ratio toward more depression with a ratio of 0.70, compared with 1.02 in control calcium (2 mM; n = 6; P < 0.01). Elevated calcium also slightly increased the amplitude of the average initial EPSC, from 114.9 pA to 143.2 pA (n = 6; P = 0.113). Under decreased calcium conditions (1 mM), the paired pulse ratio shifted toward greater enhancement, from 1.01 to 1.58 (n = 5; P < 0.01) and decreased the amplitude of the initial EPSC, from 152.9 pA to 58.4 pA (n = 5; P = 0.055). During individual recordings, paired pulse ratios could change from net enhancement to depression and vice versa.
Comparison with timing nuclei in the brain stem
To compare the short-term plasticity found in NA recordings with those in the timing nuclei, we made recordings from neurons in the cochlear nucleus magnocellularis (NM) and nucleus laminaris (NL). Synaptic responses recorded in NM neurons showed profound depression at this age, as has been shown previously () (Zhang and Trussell 1994b
). The steady state amplitude monotonically decreased with input stimulus rate (; open squares, n
= 3 NM neurons). These experimental results are similar to that predicted by a model of simple depression in NM (dashed line; see METHODS). The synapses onto NM neurons showed significantly more depression on average than those onto NA neurons at input rates 200 Hz and 250 Hz (P
< 0.05). NL neurons receive synaptic input from NM, and these synapses also show profound short-term depression (Cook et al. 2003
; Kuba et al. 2002
). We found that NM to NL synapses expressed significantly more depression than auditory nerve to NA synapses for all rates (; , open triangles; n
= 5 NL neurons; P
< 0.05). Although NA synaptic responses often showed net depression at steady state, they nevertheless showed much less depression on average than the synaptic responses recorded in NM or NL neurons, and had a distinctly different steady state synaptic transfer function.
To understand how the short-term plasticity expressed at the auditory nerve to cochlear nucleus synapse affects the transmission of spike rate information, we analyzed how the total steady state synaptic conductance is related to the input rate (Abbott et al. 1997
; Markram et al. 1998b
; Tsodyks and Markram 1997
). We used as an approximation of total conductance the product of the steady state amplitude with the presynaptic spike rate (). This calculation provides an estimate of the net impact on the postsynaptic neuron per unit time, assuming linear summation of the input conductance. For depressing synapses such as those in NM, the relationship of the total conductance with input rate is a nonlinear, saturating curve (, symbols as in ). This is because the decreasing steady state amplitude offsets the increase in input stimulus rate, keeping the total conductance nearly constant. The curve for NA was linear (; linear fit, slope = 0.50, R2
= 0.98), implying that changes in the input stimulus rate would result in a proportional change in total postsynaptic conductance.
Simulation using in vivo spike patterns shows that short-term synaptic plasticity in NA can help maintain the rate code for intensity
To better understand how short-term synaptic plasticity may affect sound coding, we simulated the synaptic driving conductance in response to input trains that had been recorded from auditory nerve fibers in vivo in hatchling chick (nerve data provided courtesy of Dr. James C. Saunders, University of Pennsylvania). The auditory nerve data consisted of spike trains recorded in response to a 40 ms tone stimulus at best frequency, across a range of sound levels (−5 to +40 dB relative to threshold), with 200 trials per level. The simulations used synaptic model parameters from fits to three representative NA synapses: two nonmonotonic (one of which is the synapse shown in , and one which had a steady-state profile similar to the average NA profile), and one monotonically depressing (synapse shown in ).
As illustrated in , each presynaptic spike (top panel
) was assumed to result in a postsynaptic conductance, simulated as an alpha-like function (middle panel
) with a time course similar to a quantal EPSC in NA (MacLeod and Carr 2005
). The amplitude of the conductance was varied according to the output of the short-term plasticity model driven by the inter-spike intervals present in the input trains. We summed the simulated conductances across the set of in vivo trials for each sound level, creating an alpha-function-smoothed poststimulus time histogram (α
PSTH; bottom panel
). The α
PSTH thus represents the input conductance versus time, summed over multiple sequential trials, or large numbers of inputs. The α
PSTHs that resulted from the simulation using each representative synapse are plotted in . To compare across intensities, the total summed conductance versus stimulus intensity (“Level” was calculated (bar plots in ), either over the whole stimulus (white bars, “0–40 ms”), or just during the tonic component (gray bars, “20–40 ms”). The spike frequency PSTHs of the original auditory nerve trains show the primary-like profile (phasic and tonic components) which is characteristic of all auditory nerve responses (). In the absence of facilitation or depression (“no plasticity”), the α
PSTH is a smoothed version of the spike frequency PSTH (). Increasing sound levels produced increasing summed conductance, reflecting the increasing pre-synaptic firing rates (). The simulated nonmonotonic NA synapse in shows that this NA synapse produced an α
PSTH very close to the “no plasticity” case, and preserved most of the intensity-related range in the total conductance during the tonic component (gray bars, ). In contrast, for the purely depressing synapse the α
PSTHs converge within 10–15 ms (). The phasic component still contains intensity information (white bars, ), but there is no information in the tonic component (gray bars, ). The results for a third NA synapse showed intermediate behavior and maintenance of some degree of intensity information during the tonic component (). The synaptic plasticity observed in NA can clearly influence how the sound level information of sound stimuli is transmitted. While depressing synapses can convey intensity information at the onset of the stimulus, the intensity of ongoing stimuli can only be conveyed by synapses that maintain the amplitude of the synaptic conductance by enlisting facilitation to balance the depression.