Distinct kinetics of synaptic structural plasticity,memory formation,and memory decay in massed and spaced learning

Long-lasting memories are formed when the stimulus is temporally distributed (spacing effect). However, the synaptic mechanisms underlying this robust phenomenon and the precise time course of the synaptic modifications that occur during learning remain unclear. Here we examined the adaptation of horizontal optokinetic response in mice that underwent 1 h of massed and spaced training at varying intervals. Despite similar acquisition by all training protocols, 1 h of spacing produced the highest memory retention at 24 h, which lasted for 1 mo. The distinct kinetics of memory are strongly correlated with the reduction of floccular parallel fiber–Purkinje cell synapses but not with AMPA receptor (AMPAR) number and synapse size. After the spaced training, we observed 25%, 23%, and 12% reduction in AMPAR density, synapse size, and synapse number, respectively. Four hours after the spaced training, half of the synapses and Purkinje cell spines had been eliminated, whereas AMPAR density and synapse size were recovered in remaining synapses. Surprisingly, massed training also produced long-term memory and halving of synapses; however, this occurred slowly over days, and the memory lasted for only 1 wk. This distinct kinetics of structural plasticity may serve as a basis for unique temporal profiles in the formation and decay of memory with or without intervals.During learning, memories are formed in a specific population of neuronal circuits and are consolidated for persistence (1, 2). These memory processes are supported by discrete subcellular events such as reversible modifications in the efficacy of synaptic transmission (3–5) or persistent structural modifications in the size and number of synaptic connections (6–8). However, how these synaptic modifications relate to the dynamics of formation and decay of memories in behaving animals remains elusive. Memory formation and its persistence are also sensitive to the temporal features of stimulus presentation, as observed in the well-known “spacing effect.” Training trials that include resting intervals between them (spaced training) produce stronger and longer-lasting memories than do the same number of trials with no intervals (massed training) (9). The spacing effect has been observed in a variety of explicit and implicit memory tasks (10–13), and the molecular mechanisms supporting this phenomenon have been reported (14–18). Various intracellular signaling molecules such as CREB (19), mitogen-activated protein (MAP) kinase (20, 21), and PKA (22, 23) underlie the spacing effect and are implicated in the remodeling of neuronal structures (23). In vitro studies showed that spaced stimuli induced the protrusion of new filopodia (20) and the recruitment of new synapses (24) in hippocampal neurons. However, despite the existence of numerous behavioral and molecular studies, no conjoint study has elucidated the synaptic correlates that underpin the expression of the spacing effect during learning. Here we studied the temporal evolution and decay of memory and its correlation with synaptic modifications during learning with distinct temporal patterns of training.We used an adaptation of the horizontal optokinetic response (HOKR), which is a simple model of cerebellum-dependent motor learning. It is a compensatory eye movement for stabilization of the visual image on the retina during horizontal motion of the surroundings. A surrounding that oscillates horizontally at a given frequency causes retinal slips in naive animals and facilitates HOKR 1 h after training (HOKR adaptation) (25–27). The amount of adaptation can be quantitatively monitored, and the flocculus (Fl), which is a phylogenetically preserved cerebellar lobule, is involved in the adaptation of the HOKR (28, 29). These features render this paradigm as an experimental model, useful for investigating neural correlates and mechanisms involved in motor learning. In a previous study, we showed that the short-term adaptation of HOKR induced by 1-h training was accompanied by a rapid and transient reduction (28%) in the number of AMPA receptors (AMPARs) in parallel fiber (PF) to Purkinje cell (PC) synapses, whereas the long-term adaptation induced by repeated 1-h training over 5 d was accompanied by a slowly developing reduction (45%) of PF–PC synapses (30). Despite recent controversies on the role of long-term depression (LTD) and a postulated role of long-term potentiation in cerebellar motor learning (31–33), this study first showed that LTD as a form of reduced AMPARs in PF–PC synapses does occur in physiological learning.In the present study, we further examined how the spacing effect is correlated with the structural plasticity in PF–PC synapses. We showed that spaced training including 1-h intervals induced stable long-lasting memories within 4 h after the training, which was accompanied by a rapid and long-lasting (>1 mo) reduction of PF–PC synapses after a transient reduction in AMPAR density and shrinkage of PF–PC synapses and PC spines. One hour of massed training also induced a gradual reduction of the PF–PC synapses, which reached the same level as that observed for the spaced training 5 d later but recovered faster within 10 d. The time course corresponded well with the slower establishment and quicker decay of long-lasting memory induced by massed training. The tight correlation observed between the structural modifications and the kinetics of long-lasting memory pinpoints the distinct temporal regulation of synaptic connections as a mechanism underlying the spacing effect.