Abstract
Purkinje cells (PC) control spike timing of neighboring PC by their recurrent axon collaterals. These synapses underlie fast cerebellar oscillations and are characterized by a strong facilitation within a time window of <20 ms during paired-pulse protocols. PC express high levels of the fast Ca2+ buffer protein calbindin D-28k (CB). As expected from the absence of a fast Ca2+ buffer, presynaptic action potential-evoked [Ca2+]i transients were previously shown to be bigger in PC boutons of young (second postnatal week) CB-/- mice, yet IPSC mean amplitudes remained unaltered in connected CB–/– PC. Since PC spine morphology is altered in adult CB–/– mice (longer necks, larger spine head volume), we summoned that morphological compensation/adaptation mechanisms might also be induced in CB–/– PC axon collaterals including boutons. In these mice, biocytin-filled PC reconstructions revealed that the number of axonal varicosities per PC axon collateral was augmented, mostly confined to the granule cell layer. Additionally, the volume of individual boutons was increased, evidenced from z-stacks of confocal images. EM analysis of PC–PC synapses revealed an enhancement in active zone (AZ) length by approximately 23%, paralleled by a higher number of docked vesicles per AZ in CB–/– boutons. Moreover, synaptic cleft width was larger in CB–/– (23.8 ± 0.43 nm) compared to wild type (21.17 ± 0.39 nm) synapses. We propose that the morphological changes, i.e., the larger bouton volume, the enhanced AZ length and the higher number of docked vesicles, in combination with the increase in synaptic cleft width likely modifies the GABA release properties at this synapse in CB–/– mice. We view these changes as adaptation/homeostatic mechanisms to likely maintain characteristics of synaptic transmission in the absence of the fast Ca2+ buffer CB. Our study provides further evidence on the functioning of the Ca2+ homeostasome.
Highlights
Activity-dependent synaptic plasticity, i.e., modulation of the synaptic strength of two neurons coupled by chemical synapses is the hallmark of most synapses (Kim and Linden, 2007)
At the end of the second PN week Purkinje cells (PC) axon collateral arbors stabilize their length, spatial distribution and number of axonal boutons, a process probably achieved by cerebellar myelination that limits the expansion of axon collaterals and conserves final and stable synapses (Gianola et al, 2003)
Semiquantification of Ca2+ buffer (CB) expression measured by the intensity of fluorescence in the PC layer indicated a developmental upregulation of CB (Figures 1A,B)
Summary
Activity-dependent synaptic plasticity, i.e., modulation of the synaptic strength of two neurons coupled by chemical synapses is the hallmark of most synapses (Kim and Linden, 2007). Time scales of hours to days and the process is referred to as HSP (Turrigiano, 2012) The latter is thought to maintain network stability by fine-tuning global synaptic strength under conditions when its activity diverges from tolerant (stable) physiological levels, e.g., as the consequence of an insult, chronic suppression of activity or mutations in genes implicated in synaptic transmission. Among other components linked to Ca2+ entry and extrusion, Ca2+ buffers are considered as relevant modulators of these presynaptic Ca2+ signals. Examples of such buffers characterized by either slow or fast Ca2+-binding kinetics include parvalbumin (PV) and CB, respectively
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