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The extent to which synaptic activity can signal a sensory stimulus limits the information available to a neuron. We determined the contribution of individual synapses to sensory representation by recording excitatory postsynaptic currents (EPSCs) in cerebellar granule cells during a time-varying, quantifiable vestibular stimulus. Vestibular-sensitive synapses faithfully reported direction and velocity, rather than position or acceleration of whole-body motion, via bidirectional modulation of EPSC frequency. The lack of short-term synaptic dynamics ensured a near perfect linear relationship between velocity and charge transfer, and as few as 100 synapses provided resolution approaching psychophysical limits. This indicates that highly accurate stimulus representation can be achieved by small networks and even within single neurons.
Sensory representation relies on the computational efficiency of individual neurons, which is limited by the amount of information available to a cell through its synaptic inputs. The quality of these synaptic signals will depend not only on presynaptic firing rates, but also on the stochastic properties and the short-term dynamics of release, postsynaptic receptor activation and desensitization. Although sensory representation has been studied in many cell types, poor control of sensory input parameters and uncertainty regarding the number of synapses involved in compound responses have hampered our understanding of unitary synaptic contribution. To overcome these problems we studied granule cells (GCs) in the cerebellar flocculus (Fig. 1A) where the contribution of individual mossy fibre (MF) inputs can be resolved (1, 2). However in contrast to previous studies (2, 3) this preparation permits the use of a highly accurate and quantifiable vestibular stimulus over a large region of sensory space (4-6). Moreover because several features of motion detection along the vestibular - cerebellar pathway have been elucidated by extracellular recordings (4-7) the synaptic information content can be placed in the broader context of cerebellar function.
In vivo whole-cell voltage clamp recordings (8) in ketamine- and xylazine-anesthetized mice (9) revealed the presence of spontaneously occurring EPSCs with a mean frequency of 13 ± 2.3 Hz (n = 18 cells) in the absence of a vestibular stimulus. During horizontal rotation (Fig. 1B), a bidirectional modulation of EPSC frequency was observed (range from 0 to 110 Hz, time bins = 100 ms) (Fig. 1C and movie S1). Plotting the EPSC frequency as a function of the stimulus parameters angular position (green), velocity (black), and acceleration (orange) revealed that EPSC rate was linearly related to velocity (r2 = 0.7 ± 0.04) but not to position (r2 = 0.05 ± 0.02) or acceleration [r2 = 0.03 ± 0.01; analysis of variance (ANOVA), P < 10−20, n = 18 cells] (Fig. 1D). Synaptic responses to vestibular stimulation fell into one of two distinct classes (Fig. S1): positive rate modulation in the ipsilateral direction and negative modulation in the contralateral direction (type 1, n = 9) or vice versa (type 2, n = 9) (5). The GC excitatory drive, as quantified from the change in total charge transferred, was also modulated in a manner similar to that of EPSC frequency (Fig. S1). Both type 1 and type 2 cells showed a near-perfect correlation between EPSC frequency and velocity (type 1: r = 0.94 ± 0.09, n = 9; type 2: r = 0.97 ± 0.01, n = 9) (Fig. 1E). Although the slope (gain) of the relationship between EPSC frequency and velocity varied widely across cells (Fig. 1E, inset), the mean gain was also similar for type 1 and type 2 responses (type 1: 0.42 ± 0.11 Hz / (°/s), type 2: 0.36 ± 0.07 Hz / (°/s); P = 0.68). However, because inputs could be silenced at high velocities in the non-preferred direction (mean decrease = 58 ± 9%, range of 0 to 100%, n = 18 cells) (Fig. 1F) the correlation between EPSC frequency and velocity deteriorated in the non-preferred direction [r(pref. direct.) = 0.952; r(non-pref. direct.) = 0.753] (Fig. 1G).
MF-GC EPSCs have been shown to exhibit frequency-dependent depression (10, 11) and glutamate-spillover-mediated AMPA receptor activation (12), suggesting that the excitatory charge in vivo could be a nonlinear function of velocity. We therefore assayed the synaptic excitatory drive by quantifying the amplitude and decay time course of spontaneous and motion-evoked EPSCs (Fig. 2A). In the majority of cases (n = 13/18 cells) we observed no significant difference (P > 0.05) in EPSC amplitude or EPSC weighted decay between spontaneous EPSCs and those recorded during movement in the preferred direction (Fig. 2B). Moreover, there was no change in the EPSC amplitude distribution as the angular velocity was increased in the preferred direction (Fig. 2, C to E). The relationship between charge per unit time, (calculated over 100 ms bins) and both EPSC frequency (r = 0.78 ± 0.05, n = 18 cells) and motion velocity (r = 0.73 ± 0.06, n = 18 cells) was linear (Fig. 2, F and G). This lack of short-term dynamics over these EPSC frequencies therefore allows angular velocity to be linearly represented both by EPSC frequency and excitatory charge at the postsynaptic membrane. Furthermore the coefficient of variation of EPSCs observed in this class of responses (cv = 0.55 ± 0.03, n = 13 cells) was similar to that for single MF inputs recorded in vitro (12), whereas the frequencies of both spontaneous and evoked EPSCs observed here are entirely consistent with recordings from individual MFs showing high action potential rates in vivo (5, 11, 13) (Fig. S2).
Some GCs (n = 5/18 cells) (Fig. 2A, solid circles) did, however, exhibit a significant increase in EPSC amplitude in the preferred direction (P < 0.05; Fig. 3,A and B) and a significantly higher cv (0.67 ± 0.04, P < 0.05). In these cells, the cumulative amplitude histogram changed shape during motion in the preferred direction, allowing in three out of five cells the separation of distinct inputs with use of an amplitude threshold criterion (−18 to −20 pA; n = 3 cells) (Fig. 3C) (2). We always observed that, although one population of EPSCs was insensitive to our stimulus, the other population was modulated by horizontal rotation (Fig. 3D). In two out of three cells, these two EPSC populations also showed significantly different decay time courses (P < 0.05), confirming that they arose from distinct synaptic inputs. In these cases, neither the non-modulated (input 1) nor modulated (input 2) input showed frequency-dependent changes in EPSC amplitude (input 1: P = 0.99; input 2: P = 0.07, ANOVA) (Fig. 3E). Because the slow decay time (overall wdinput 1 = 3.97 ± 1.24 ms versus wdinput 2 = 3.28 ± 1.07 ms, n = 3 cells) (Fig. 3F) and high spontaneous frequency (> 7 Hz, 3 cells) of the non-modulated input are distinct from the low frequency, very fast decaying miniature events observed in vitro (14), the non-modulated population consists predominantly of action potential-evoked events. Although we cannot distinguish intrinsic from extrinsic MF-evoked EPSCs, the overall similar decay time course (P = 0.17 n = 3 cells) of the non-modulated EPSC to the modulated EPSC and those EPSCs evoked by stimulation of a single extrinsic MFs in vitro (12) suggests that they arise from the same class of MFs.
Because it is necessary for individual and populations of synapses to reliably report stimulus information online during a sensory event, coherent representations in the cerebellum are likely to involve signals distributed over many MF-GC connections. We used a Bayesian reconstruction algorithm (9) (Fig. S3) to quantify the ability of the MF-GC synapse to report our motion stimulus. Although activity through a single synapse could indicate the direction of motion (Fig. 4A), its estimate of motion velocity (per time bin) suffered from significant errors but improved with increasing synapse number towards psychophysical estimates (err1 synapse = 15.6 ± 1.3 °/s versus err100 synapses = 4.8 ± 0.5 °/s, stimulus range from −37.7 to +37.7 °/s, P < 10−8) (Fig. 4, B and C). In parallel, the reliability (SD of error) of the velocity estimate significantly improved with the logarithm of synapse number (r = −0.998, P < 0.01) (Fig. 4, B and C). This indicates that activity through as few as 100 MF-GC synapses can reliably report the direction and velocity of our motion stimulus.
We recorded motion-evoked synaptic currents and found that velocity information is represented linearly via bidirectional modulation of EPSC frequency and charge around a tonically active vestibular input. By using differences in the EPSC waveform as a signature of distinct MF-GC synapses, we also found that floccular GCs receive inputs with unique receptive fields which suggests that vestibular, visual (15), and/or eye movement - related signals (16) may converge on individual GCs (17). In contrast to a single vestibular primary afferent which can accurately report subtle changes in velocity over a single trial (18), about 100 MF-GC synapses were required to provide enough resolution to meet psychophysical predictions (18, 19). This has two major implications for information processing in the cerebellum. MF-GC synapses do not simply relay peripheral spike rates (2, 11, 18, 20) but appear to present vestibular information to be integrated with other stimulus features. Second, although the quality of velocity information may be diluted by the probabilistic nature of transmitter release and action potential firing along the sensory pathway (21), sensory information is preserved by a population synaptic signal arising from many MFs. Although the absolute number of MF-GC synapses required will depend on the time frame of cerebellar operation it seems likely that reliable velocity information must be presented to downstream Purkinje cells as an orchestrated coincident GC signal (22, 23).
The highly linear synaptic properties and broad gain distribution of vestibular inputs onto GCs provide a system well suited for cue combination tasks, where stimulus probability distributions are inferred from linear combinations of neural activity (24). The very limited number of inputs onto GCs has allowed us isolate and monitor stimulus-evoked EPSCs arising from distinct synaptic contacts (2). However in more complex neurons such as Purkinje and pyramidal cells, non-linear interactions between synaptic inputs within the dendritic tree may affect on the processing of such information at the soma (25). However, in such cells, more reliable stimulus reconstruction and multimodal processing is likely to be achieved through the integration of large numbers of inputs with similar receptive fields.
We thank R. Callister and A. Brichta for advice and helpful discussion and the members of the lab and T. Mrsic-Flögel for comments on the manuscript. This work was supported by the Wellcome Trust (A.A., R.A.S, and T.W.M.), the UK Biotechnology and Biological Sciences Research Council (A.T.S., R.A.S., and T.W.M.), and the Human Frontier Science Program (T.W.M.).