Distinct cerebellar engrams in short-term and long-term motor learning

Abstract

Cerebellar motor learning is suggested to be caused by long-term plasticity of excitatory parallel fiber-Purkinje cell (PF-PC) synapses associated with changes in the number of synaptic AMPA-type glutamate receptors (AMPARs). However, whether the AMPARs decrease or increase in individual PF-PC synapses occurs in physiological motor learning and accounts for memory that lasts over days remains elusive. We combined quantitative SDS-digested freeze-fracture replica labeling for AMPAR and physical dissector electron microscopy with a simple model of cerebellar motor learning, adaptation of horizontal optokinetic response (HOKR) in mouse. After 1-h training of HOKR, short-term adaptation (STA) was accompanied with transient decrease in AMPARs by 28% in target PF-PC synapses. STA was well correlated with AMPAR decrease in individual animals and both STA and AMPAR decrease recovered to basal levels within 24 h. Surprisingly, long-term adaptation (LTA) after five consecutive daily trainings of 1-h HOKR did not alter the number of AMPARs in PF-PC synapses but caused gradual and persistent synapse elimination by 45%, with corresponding PC spine loss by the fifth training day. Furthermore, recovery of LTA after 2 wk was well correlated with increase of PF-PC synapses to the control level. Our findings indicate that the AMPARs decrease in PF-PC synapses and the elimination of these synapses are in vivo engrams in short- and long-term motor learning, respectively, showing a unique type of synaptic plasticity that may contribute to memory consolidation.

Keywords: Golgi staining; high-voltage electron microscope; long-term depression.

Fig. 1.

HOKR training induces short- and long-term adaptation. (A) Representative eye-movement traces of a mouse on days 1 (D1) and 5 (D5), before (pre) and after (post) HOKR training. (B) Representative learning curves from mice showing only STA (#1, black), both STA and LTA (#2, blue), or no (#3, red) adaptation at all. (C) Daily STA was induced by 1-h HOKR training (*P < 0.05, **P < 0.01, vs. gain measured before the training on each day, paired t test) and LTA gradually developed over 5 d of training (^#P < 0.05 vs. gain measured before the training on day 1, paired t test). Data were presented as mean ± SEM.

Fig. 2.

STA is accompanied with rapid and transient reduction in the density of PF–PC synaptic AMPA receptors selectively in the FL. (A and B) Labeling for GluD2 subunits (15-nm gold) and AMPARs (5-nm gold) on freeze-fracture replicas from the FL of mice pre- (A) and post- (B) 1-h training. (Scale bar, 100 nm.) (C–F) Data from representative mice before and after 1-h training showed that STA was accompanied with decreased synaptic AMPAR density in the FL (C) but not PFL (E), and left-shifted distribution of AMPAR densities in the FL (D, two-sample Kolmogorov–Smirnov test: z = 1.863, P = 0.002) but not PFL (F, z = 0.930, P = 0.353). The STA did not change the positive correlation between number of AMPARs and size of postsynaptic area (C and E). (G) Pooled data indicated that the STA was accompanied with decreased AMPAR density in the FL (P = 0.036) but not PFL. No change in GluD2 receptor density was detected in the FL. (H) Data from individual mice at day 1 revealed that the changes in AMPAR density in the FL (normalized to that in the PFL) after 1-h training is negatively correlated with changes in gain (Δgain normalized to pretraining gain, Pearson correlation: R = 0.667, P = 0.002). (I) Density of synaptic AMPARs in the FL significantly decreased after 1-h training on day 1 (D1) and recovered after 24 h (D2). Although density of synaptic AMPARs in the FL also decreased significantly after 1-h training on day 3 (D3), it did not change on day 5 (D5). Black filled circles in C–F and I indicate data obtained before daily training (pre). Red open circles in C–F and I indicate data obtained after daily training (post). Data were presented as mean ± SEM, *P < 0.05, **P < 0.01 vs. D1 pre, Student t test.

Fig. 3.

LTA is accompanied with persistent but reversible elimination of the target PF–PC synapses. (A) PF–PC synaptic length was not changed at posttraining on day 1 (D1 post) or 5 (D5 post) compared with basal level (D1 pre) in either the FL or PFL. (B) The synaptic density was not changed at posttraining on day 1 (D1 post) or pretraining on day 2 (D2 pre) but decreased by 33% on day 5 (D5 post) compared with D1 pre in the FL. No change was observed in PFL. (C) Volume of the molecular layer in the FL was slightly but significantly decreased on day 5 (D5 post) compared with D1 pre. (D) Daily HOKR training gradually induced LTA which disappeared after 14 d of light-dark cycle exposure (black circles). PF–PC synaptic density showed correlated reduction in the FL (red circles) throughout this time course. The synaptic density in PFL (blue circles) remained unchanged. HOKR gain is expressed as ratio to control at pretraining. (E) Schematic drawing of representative sections of the rostral, middle, and caudal FL showing positions of EM sections used for mapping (shown in F and G) of synaptic density. The sections were sampled near the surface (dark pink) of the ML (M, light pink). The granule cell layer (G) and white matter (W) are indicated in orange and blue, respectively. Fissure between the FL and PFL is indicated with blue line (F). (Scale bar, 200 μm.) (F and G) Mapping analysis of synaptic density in the FL of control (Cont FL) and 5-d trained (D5 FL) mice showed significant reduction of synaptic density (using corresponding nine adjacent values in two groups, paired t test, P < 0.001) throughout the medio–lateral extent of the middle but not rostral and caudal FL. Area used for the physical dissector analysis (light blue) showed a prominent reduction in synapse density at D5 compared with control. Data were presented as mean ± SEM, *P < 0.05, **P < 0.01, vs. D1 pre, Student t test.

Fig. 4.

LTA is accompanied with elimination of PC spines in the FL. (A) Representative high-voltage EM images of PC spines along individual dendritic segments in the FL and PFL of control (D1 pre) and trained (D5 post) mice. (Scale bar, 2 μm.) (B) Pooled data showed selective spine elimination by 30% in the FL but not in PFL on day 5. Data were presented as mean ± SEM, **P < 0.01, vs. D1 pre, Student t test.