In this study, auditory and trigeminal synapses in the PnC were stimulated presynaptically by applying short, high frequency bursts to afferent fibers of secondary neurons in the respective sensory pathway. We chose the burst stimulus paradigm as a model for the in vivo activity of auditory neurons during an acoustic startle experiment. Primary and secondary auditory neurons have been shown to fire at high frequency during sound presentation [29–31] and our burst duration resembles the duration of a startle stimulus. Giant neurons have a slow membrane time constant, enabling them to integrate repetitive auditory inputs as well as other sensory or modulatory input arriving at the neuron at different latencies . Presynaptic bursts of action potentials produce compound synaptic currents whose amplitudes determine the probability that spiking will occur and influence the number of action potentials generated by PnC giant neurons . Such compound excitatory synaptic currents (cEPSCs) are therefore a decisive factor for the elicitation and amplitude of a startle response.
In the first two experiments, we showed that in C57/Bl6 mice (and to some extent in rats) both the tactile and acoustic startle responses decreased when repetitive stimuli of these modalities were presented and similarly demonstrated that synaptic depression is induced when acoustic or trigeminal fibers synapsing on PnC giant neurons are repeatedly stimulated.
There was a slight difference in absolute acoustic startle amplitudes in one group of animals tested before tactile habituation (Fig. 4A, block 1–10) and in two other groups of animals tested either after tactile habituation or after exposure to the 95 dB background noise only (Fig. 4B, block 11–15). This difference was small (180 and 150 mV, respectively) and statistically insignificant. The 95 dB background noise used during tactile stimulation could account for the slightly lower acoustic startle amplitudes in Fig. 4B. However, after exposure to background noise, both groups of animals in Fig. 4B still showed acoustic startle habituation not statistically different from the habituation in Fig. 4A. The critical factor in this experiment is the lack of difference between the acoustic startle amplitudes of the two groups of mice in Fig. 4B (blocks 11–15, t-values < 1), even though only one group had received prior tactile stimulation.
Interestingly, the decline in startle response amplitudes of the C57BL/6 mice was generally greater when the tactile (behavioral) or trigeminal (electrophysiological) afferents were stimulated than when acoustic/auditory fibers were stimulated. This implies that short-term habituation to acoustic stimuli is generally weaker in these animals compared to habituation to tactile stimuli. Indeed, it has been reported that the amount of short-term habituation varies enormously between different stimulus modalities and that the degree of habituation is also dependent on the mouse strain . However, we cannot completely exclude the possibility that habituation to the (perhaps fear and/or stress-inducing) background noise during tactile startle measurements summates with the tactile startle habituation, although the background noise was switched on 5 min. before measurements started and tactile habituation showed a perfect exponential decay. Moreover, background noise should actually lead to sensitization, a process that counteracts habituation . Additionally, the behavioral results closely parallel the electrophysiological findings, adding to the growing body of evidence that PnC giant neurons are indeed a vital station in the startle pathway [13, 16, 18–20]. The results also strongly indicate that habituation takes place inside this pathway, namely in the form of synaptic depression at the synapse of the sensory inputs to the giant neurons [5, 11, 25–28].
Localization of synaptic depression
The main goal of this study was to determine whether the cellular substrate for short-term habituation, which is presumably the result of synaptic depression in sensory synapses within the PnC, is located in the sensory part of the startle pathway (i.e. at the presynaptic terminals of sensory afferents), or if this is instead a postsynaptic feature. Given the association of synaptic depression with short-term habituation, a presynaptic mechanism would imply that short-term habituation occurs before signals from different pathways are integrated in the PnC. Habituation would therefore be specific for each stimulus modality and would not generalize between different modalities. We tested this in electrophysiological experiments with rats and mice and in behavioral experiments conducted with mice from the same strain (i.e. C57BL/6 mice, also commonly used to generate knock-out mice). This is vital, as large differences between different mouse strains have been found with respect to habituation [7, 8].
Both approaches showed that both synaptic depression and short-term habituation are pathway specific. This is also consistent with behavioral studies of other species and mouse strains, which demonstrated that short-term habituation is modality specific [7, 34]. The pathway specificity signals that a presynaptic mechanism is involved and further evidence is provided by our data showing that the paired pulse ratio changes during depression, since alterations in short-term plasticity are commonly thought to reflect presynaptic changes in the transmitter release machinery [34, 35]. However, it should be noted that the paired pulse ratio declined during synaptic depression, which is the opposite of what would be expected if the decrease in synaptic efficacy is due to a reduced probability of transmitter release. This is normally accompanied by an increase in the paired pulse ratio, since the accumulated calcium in the presynaptic terminal meets a larger pool of releasable transmitter [35, 36].
Different mechanisms could account for a reduced paired pulse ratio; either the desensitization of postsynaptic glutamate receptors or presynaptic mechanisms, such as the exhaustion of releasable transmitter in the presynaptic terminal.
Mechanism of depression
Only repeated activation by short trains of at least four presynaptic action potentials produced an exponential decay of the cEPSC amplitudes in PnC neurons. Since trains of presynaptic action potentials lead to a strong and sustained release of the neurotransmitter glutamate into the synaptic cleft, desensitization of postsynaptic glutamate receptors could easily account for the resulting synaptic depression. However, several results rule out receptor desensitization as the underlying mechanism; first, the time course and the degree of depression were comparable when bursts were applied either every second or only every 10 seconds. In contrast, recovery from receptor desensitization should occur completely within 10 sec. Second, synaptic responses to single pulses as well as the responses to the first pulse within a burst were not (or not equally) affected by synaptic depression, but desensitized postsynaptic receptors should have had a similar effect on both response types. Moreover, repeated uncaging of exogenous glutamate at 1 Hz evoked responses with no sign of desensitization.
In summary, all of our results clearly contradict the hypothesis that postsynaptic receptor desensitization or changes in postsynaptic passive conductances can account for synaptic depression. Instead, our data provide ample behavioral and electrophysiological evidence that synaptic depression is a presynaptic phenomenon. Future studies will have to identify the molecular processes responsible for the alterations in synaptic transmission described here.