A stable proportion of Purkinje cell inputs from parallel fibers are silent during cerebellar maturation

Cerebellar Purkinje neurons integrate information transmitted at excitatory synapses formed by granule cells. Although these synapses are considered essential sites for learning, most of them appear not to transmit any detectable electrical information and have been defined as silent. It has been proposed that silent synapses are required to maximize information storage capacity and ensure its reliability, and hence to optimize cerebellar operation. Such optimization is expected to occur once the cerebellar circuitry is in place, during its maturation and the natural and steady improvement of animal agility. We therefore investigated whether the proportion of silent synapses varies over this period, from the third to the sixth postnatal week in mice. Selective expression of a calcium indicator in granule cells enabled quantitative mapping of presynaptic activity, while postsynaptic responses were recorded by patch clamp in acute slices. Through this approach and the assessment of two anatomical features (the distance that separates adjacent planar Purkinje dendritic trees and the synapse density), we determined the average excitatory postsynaptic potential per synapse. Its value was four to eight times smaller than responses from paired recorded detectable connections, consistent with over 70% of synapses being silent. These figures remained remarkably stable across maturation stages. According to the proposed role for silent synapses, our results suggest that information storage capacity and reliability are optimized early during cerebellar maturation. Alternatively, silent synapses may have roles other than adjusting the information storage capacity and reliability.

Typical central excitatory synapses are formed onto dendritic spines, the distinctive morphology of which enables their unambiguous identification (1–3). It has generally been assumed that the presence of a dendritic spine equates to the existence of a functional excitatory transmission (4). Based on this assumption, the observation of spine motility in several brain areas (motor cortex and somatosensory cortex) has been considered to reflect synaptic plasticity (5). Indeed, a number of studies have established a correlation between learning and spine formation (6–8) or pruning (9, 10). However, in some conditions, morphological and synaptic plasticity have been shown to be dissociated (11), in line with the view that morphology does not provide all the information necessary to infer synaptic function.The cerebellum contains the majority of brain neurons (12, 13) and the predominant excitatory synapses found in this structure connect granule cells (GC) to Purkinje cells (PC). These synapses are formed on typical spines borne by PC dendrites (14), the majestic shape of which is likely related to the huge amount of independent inputs they receive. The GC-to-PC synapse is generally acknowledged to be an essential site for plasticity (15–19). However, in sharp contrast to other parts of the brain such as motor and somatosensory cortices, PC spines appear to be constitutive (20, 21), i.e., they appear to be an inherent property of PCs, independent of external factors. Indeed, pruning of these synapses has not been reported. Novel spine formation has been reported, but likely as a result of dendritic tree expansion (22, 23). The high density of spines along PC dendritic branchlets (5 to 17 per linear micrometer in rat; refs. 14 and 24–27) and their regular ordering in a helical pattern (28) support the idea that they optimize space occupancy with little room for spine addition, in accordance with their constitutive nature.The apparent morphological homogeneity of PC spines is in sharp contrast with the spectacular heterogeneity observed in the strength of GC-to-PC synapses. An in vivo study has reported that the receptive field of a PC was much smaller than that of the GCs putatively connecting to it (29), suggesting that most GC-to-PC synapses are electrically silent. This has been confirmed by an in vitro report (30) showing that synaptic transmission between paired-recorded GC and PC was detected nearly 10 times less frequently than expected from the occurrence of morphologically defined synaptic connections predicted by anatomical data (14, 31–33). Taken together, these two studies conducted in adult rats suggest that most (85% according to ref. 30) morphologically and molecularly defined GC-to-PC synapses are silent, i.e., they do not transmit any detectable electrical signal.If silent synapses do not transmit information, what is their role? Are they a reserve for additional information storage? Or do they result from information storage optimization (34)? According to this latter proposal (17, 34), since the requirement for optimized information storage is more and more critical as the amount of learned information increases, one might expect that the proportion of silent synapses increases with the amount learning. As previously suggested (34), this hypothesis could be tested by comparing the proportion of silent synapses in young versus adult animals. Indeed, the mouse cerebellar circuitry is not fully in place until the third postnatal week. Then, for at least 3 wk, the mouse acquires basic skills (eating, walking, and social interactions), adapts to changes in muscle strengths and sensitivity to stimuli, and improves its agility (35). Although the amount of cerebellar learning occurring over this maturation period is unclear, it can be reasonably assumed that cerebellar operation continuously optimizes. Here, we investigate how the proportion of silent synapses changes over this period of maturation.We determine the proportion of silent GC-to-PC synapses by a method based on the determination of the average postsynaptic response per activated synapse (average synaptic weight, w¯) in superfused acute slices. Thanks to the geometrical and repetitive architecture of the cerebellar cortex, calcium imaging is used to quantitatively map GC inputs. This mapping, combined with postsynaptic recording of transmission, and the determination of two cerebellar anatomical features (the average synapse density and spacing between PC planar dendritic trees) enables the determination of w¯. By comparison with the properties of synapses that produce an electrical postsynaptic response (investigated by paired recording and quantal analysis), we show that the proportion of silent synapses is higher than 70% and stable between the postnatal stages of interest. This suggests that cerebellar maturation has insignificant impact on the proportion of silent synapses.