Auditory and trigeminal inputs have been shown to converge on PnC giant neurons (see Fig. 1) and both undergo homosynaptic depression when repeatedly stimulated by presynaptic bursts [22, 28]. To induce synaptic depression, auditory and trigeminal afferent fibers were stimulated with 150 μs pulses applied by concentric extracellular electrodes placed either in the auditory stria ventral to the lateral superior olive (LSO, Fig. 2A) or medial to the ventral portion of the principal nucleus of the 5th nerve (Pr5, Fig. 2A) in rat and mouse brain slices. Four stimulus pulses delivered within 12 ms made up each burst and 100 bursts were applied at frequencies of either 1 or 0.1 Hz. The burst stimulation elicited short trains of action potentials in the afferent sensory fibers that should resemble the normal neuronal activity (high frequency firing) in these fibers during the presentation of an acoustic or tactile startle stimulus. The acoustic stimuli used in behavioral studies are typically sudden, loud noise pulses (>80 dB SPL) of about 10 to 30 ms duration and presented at frequencies between 0.1 and 0.03 Hz.
The postsynaptic currents evoked by the single pulses within one burst summated strongly due to the large membrane time constant of giant neurons. Thus, burst stimulation of sensory afferents evoked compound synaptic responses in PnC neurons (see Fig. 2B) that have been shown to be mediated by ionotropic glutamate receptors [28]. Peak amplitudes of the compound postsynaptic excitatory currents (cEPSCs) were measured with whole-cell patch clamp recordings at -70 mV. During a sequence of 100 bursts applied to sensory afferent fibers at a frequency of 1 Hz, the cEPSC peak amplitudes decreased to 56 ± 3 % of the initial response evoked when auditory fibers were stimulated (means of cEPSC amplitudes 91–100, n = 29) and dropped to 60 ± 2 % of the initial amplitude when trigeminal fibers were stimulated (n = 44, see Figs. 2B and 2C and). The depression of the cEPSCs lasted for about 10 minutes before amplitudes recovered to the initial value (data not shown, see [22, 28]). Resting membrane potentials and input resistances of the postsynaptic neurons were analyzed during the recordings. The resting potential was -54.7 ± 1.53 mV before and -53 ± 1.42 mV immediately after depression of burst responses was induced by 100 auditory burst stimuli at 1 Hz (n = 17). The input resistance of the recorded cells was 100 ± 17 MΩ before and 114 ± 19 MΩ during depression (n = 22), as assessed by the I/O function of current responses to three small hyperpolarizing voltage pulses. This shows that the postsynaptic cell parameters were quite stable during recordings and that there were no alterations in postsynaptic membrane permeability at or near rest during depression.
Interaction of auditory and trigeminal input in the PnC
If synaptic depression is due to a presynaptic mechanism located at the sensory afferent neuron terminals, synaptic depression in one sensory pathway should not affect synaptic transmission in the other pathway. We tested this hypothesis in rats and C57BL/6 mice. Presynaptic stimulation intensity was adjusted until similar cEPSC amplitudes were elicited by stimulation in either pathway. 100 bursts were first applied to the trigeminal pathway and then immediately thereafter to the auditory afferent fibers. Figure 3 (top and middle) shows that repeated trigeminal stimulation in rats and mice induced an exponential decay of cEPSC amplitudes (sequence 1). The auditory stimulation following again led to an exponential decay (sequence 2). In mice, we additionally reversed the order of the pathway stimulation after a 15 min recovery period (bottom, sequences 3 and 4). In summary, each 100 burst sequence induced a significant cEPSC decay (general linear model: F(1,490–881) ≥ 21.49, p < 0.0001). Moreover, there was no significant difference between the first trigeminal cEPSCs of sequences 1 and 3 or between the first auditory cEPSCs from sequences 2 and 4 (t(4) ≤ 0.035, p ≥ 0.48), indicating that synaptic depression in one pathway inhibited neither synaptic transmission in the other pathway nor subsequent synaptic depression.
Interaction of acoustic and tactile startle habituation
Given that synaptic depression is the mechanism underlying behavioral short-term habituation, habituation should also be specific to the modality used to induce it. In other words, habituation to acoustic stimuli should not affect the startle response to tactile stimuli, and vice versa. We tested this hypothesis in C57/BL6 mice. In C57/BL6 mice, both the acoustic and tactile startle responses decreased significantly (Fig. 4, blocks 1–10; general linear model: F(1,107) ≥ 8.92, p ≤ 0.0035). However, habituation to tactile startle stimuli was much stronger than habituation to acoustic stimuli in this mouse strain. In both cases, habituation had no influence on the startle response to subsequent stimuli of a different modality. The tactile startle response was the same in the group with preceding acoustic stimulation (group "acoustic→tactile") and the control group with no previous stimulation (group "tactile only"; block 11 difference: t(22) = 0.31, p = 0.76; difference of means of blocks 11–15: t(22) = 1.84, p = 0.080; Fig. 4). The course of the acoustic startle response was also the same whether or not tactile stimuli were presented before the acoustic stimuli (block 11 difference: t(22) = 0.22, p = 0.83; difference of means of blocks 11–15: t(22) = 0.07, p = 0.94).
In summary, the data shows that both synaptic depression and short-term habituation are pathway/modality specific. This also corroborates the hypothesis that short-term habituation of the startle response is associated with synaptic depression in the PnC.
Depression of burst responses is accompanied by altered short-term plasticity
The pathway specificity of synaptic depression may indicate that a presynaptic mechanism is the underlying factor. However, a local postsynaptic mechanism restricted to the activated postsynaptic sites (e.g. glutamate receptor desensitization) could also account for pathway specificity. A feature that is commonly associated with presynaptic modulation of synaptic efficacy is an alteration of short-term plasticity. In the following experiment, we used a common paired-pulse paradigm to test whether short-term plasticity changed during synaptic depression. Pairs of pulses with an interstimulus interval (ISI) of 50 ms were applied before (control) and immediately after a sequence of 100 bursts (depressed, HSD). The single EPSCs evoked by each of the pulses were clearly separated from one another (Fig. 5A). Only ten traces were averaged for each condition to ensure that synapses in the "depressed" state could not recover during paired pulse measurements. There was little variability in the EPSC amplitudes within one condition and the amplitude of EPSC1 did not change during 10 repetitions of the paired pulse protocol (1st versus 10th EPSC1: t(15) = 0.49, p = 0.315), showing that paired pulses themselves did not induce synaptic depression. The paired pulse ratio was determined for each cell from the averaged trace. Under control conditions, EPSC2 amplitudes were always substantially larger than EPSC2 amplitudes by a mean factor of EPSC2/EPSC1 = 1.83 ± 0.16 (n = 17 cells, Fig. 5A). Surprisingly, the mean amplitude of EPSCs1 in the depressed state directly after the application of 100 burst stimuli did not decrease (-116.6 ± 28.3 pA before and -122.7 ± 33.1 pA during depression, n = 17, Fig. 5B), whereas the mean amplitude of the second EPSCs decreased from -184.9 ± 42.7 pA to -160.8 ± 43.4 pA, revealing a significantly reduced mean paired pulse ratio of EPSC2/EPSC1 = 1.36 ± 0.08 during depression (t(16) = 3.36, p = 0.002, Fig. 5c). In summary, this shows that paired pulses themselves did not induce synaptic depression and that the depression induced by 100 bursts was accompanied by alterations in the paired pulse ratio. Moreover, responses to single pulses did not seem to be depressed after 100 bursts, instead the alteration in short-term plasticity as assessed by the reduced paired pulse facilitation may account for much of the attenuation of the compound EPSC amplitude resulting from a burst stimulus. In other words, the above experiments indicate that the depression of cEPSC amplitudes is not due to general depression of synaptic transmission, but is instead the result of alterations in short-term plasticity that specifically lead to an attenuation of responses to stimuli late within a burst. We verified this by analyzing the burst responses evoked by the 100 bursts in more detail. The amplitude of the first response within one burst was measured in all cells with clearly distinguishable single responses within one burst (see inset, Fig. 5D). It was found that the first responses were not as strongly depressed as the overall cEPSC amplitudes. In figure 5D, the cEPSC amplitudes and the amplitudes of the initial response within each burst are plotted for each pathway. In 30 cells the first response declined to 71% of the starting amplitude at auditory synapses but to only 81% at trigeminal synapses, whereas the overall depression of cEPSCs in the same cells was 61% in both pathways. Moreover, depression of the first response started only after about 20 bursts, indicating that short-term depression within one burst increases with repeated burst stimulation and that this accounts for the majority of the attenuation of cEPSC amplitudes.
Multiple stimuli at high frequency are necessary to induce synaptic depression
As reported above, the application of paired pulses induced no synaptic depression but burst stimulation did. We thus examined in more detail which type of stimulus is required to induce synaptic depression. We truncated the bursts to only one, two or three pulses to see whether multiple high frequency stimuli are necessary to induce depression in the auditory pathway. Application of 100 single or double pulses with 4 ms ISI at 1 Hz did not yield any synaptic depression (Fig. 6A). There was no significant difference between the amplitudes of the first and the last ten averaged EPSCs (t(18) = 0.6, p = 0.27 for single pulses; t(20) = -0.9; p = 0.18 for double pulses, Fig. 6A). Stimulation with 100 triple pulses first led to a small potentiation, followed by a significant decay of response amplitudes to 93 ± 3 % (t(18) = 6.8; p < 0.0001, Fig. 6A). To summarize, synaptic depression strongly depended on repetitive stimulation by bursts; only bursts containing three or more pulses were able to induce synaptic depression.
Evidence for a presynaptic mechanism
The decrease in paired pulse ratio during depression and the need for repeated high frequency stimulation to produce depression both indicate that postsynaptic receptor desensitization may play a role in synaptic depression. If synaptic depression is indeed caused by receptor desensitization, longer intervals between bursts should decrease the amount of depression should be weaker with longer burst intervals; we therefore, we increased the interval between the bursts from one to ten seconds in another experiment. 100 bursts applied to auditory afferents at 0.1 Hz resulted in synaptic depression of the burst responses, reducing them to 64 ± 6 % (n = 8) with a course of depression similar to that induced by 1 Hz burst stimulation (Fig. 6B). There was no significant difference between the means of EPSCs 91–100 with stimulation at 1 Hz and 0.1 Hz (t(40) = 0.58, p = 0.56), which indicates that synaptic depression was independent from the burst intervals used. The degree of synaptic depression induced by different stimulus paradigms is summarized in figure 6C. Although the results again indicated that synaptic depression is the result of a presynaptic process, we wanted to look for more evidence that could exclude receptor desensitization as the mechanism underlying the depression. We combined presynaptic stimulation of auditory afferent fibers with the application of exogenous glutamate to PnC giant neurons in rat brain slices; the glutamate was delivered via focal glutamate uncaging, thus circumventing the presynaptic terminal. We adjusted the amount of glutamate uncaging in such a way that glutamate-evoked currents displayed time courses comparable to presynaptically evoked cEPSCs (Fig. 7A). The experiment revealed two main results; first, a sequence of 100 applications of exogenous glutamate at 1 Hz did not result in a decrease of glutamate-evoked current amplitude (-134.5 ± 42.1 pA for the first and -133.5 ± 43.8 pA for the 100th glutamate-evoked current amplitude, t(8) = 1.24, p = 0.125, Fig. 7B). This indicates that repeated activation of glutamate receptors on PnC giant neurons at 1 Hz does not lead to receptor desensitization. Second, glutamate-evoked currents (GECs) in PnC giant neurons were measured before and immediately after depression was induced with a sequence of 100 burst stimuli applied to auditory afferent fibers. The amplitude and time course of the glutamate-evoked currents did not change during synaptic depression (-34.4 ± 9.8 pA before and -35.9.9 ± 9.8 pA after induction, t(10) = -1.4, p = 0.19, Fig. 7C), suggesting that no glutamate receptor desensitization occured. In summary, our data provided no evidence that postsynaptic glutamate receptor desensitization is the mechanism underlying synaptic depression in the PnC.
Further evidence for a presynaptic depression mechanism was provided by experiments in which β-GDP (2 mM) was added to the patch pipette solution. β-GDP deactivates postsynaptic G-proteins upon diffusion into the recorded cell. The effect of β-GDP is especially interesting, since there is evidence for the involvement of metabotropic glutamate receptors (probably of group III) in the synaptic depression process [28]. The addition of β-GDP had no effect on synaptic depression in the auditory or trigeminal pathway; cEPSC amplitudes elicited by auditory or trigeminal afferents significantly decreased to 50 ± 3 % (auditory) and 58 ± 3 % (trigeminal) of the initial amplitude (general linear model, F(1,784) = 412, p < 0.0001 for auditory and F(1,791) = 439, p < 0.0001 for trigeminal cEPSCs, Fig. 8). The results show that no G-protein activation at postsynaptic sites is required for synaptic depression.
As a whole, our results provided an abundance of evidence that presynaptically-located synaptic depression in the PnC is the mechanism responsible for short-term habituation of the startle response.