Effect of Associative Learning on Memory Spine Formation in Mouse Barrel Cortex

Abstract

Associative fear learning, in which stimulation of whiskers is paired with mild electric shock to the tail, modifies the barrel cortex, the functional representation of sensory receptors involved in the conditioning, by inducing formation of new inhibitory synapses on single-synapse spines of the cognate barrel hollows and thus producing double-synapse spines. In the barrel cortex of conditioned, pseudoconditioned, and untreated mice, we analyzed the number and morphological features of dendritic spines at various maturation and stability levels: sER-free spines, spines containing smooth endoplasmic reticulum (sER), and spines containing spine apparatus. Using stereological analysis of serial sections examined by transmission electron microscopy, we found that the density of double-synapse spines containing spine apparatus was significantly increased in the conditioned mice. Learning also induced enhancement of the postsynaptic density area of inhibitory synapses as well as increase in the number of polyribosomes in such spines. In single-synapse spines, the effects of conditioning were less pronounced and included increase in the number of polyribosomes in sER-free spines. The results suggest that fear learning differentially affects single- and double-synapse spines in the barrel cortex: it promotes maturation and stabilization of double-synapse spines, which might possibly contribute to permanent memory formation, and upregulates protein synthesis in single-synapse spines.

Figure 1

3D serial section EM reconstruction of three spine types from B2 barrel hollow, also shown in single electron micrographs: sER-free spine (a), spine containing sER (b), and spine containing spine apparatus (c). White arrows indicate sER ((b) and (c)) and black arrow indicates spine apparatus (c). (d)–(f) show reconstruction of dendritic spines (blue): excitatory synapses (green), inhibitory synapse (red; only (e)), smooth endoplasmic reticulum (yellow; (e)), and spine apparatus (red; (f)). Scale bars: 0.5 μm.

Figure 2

Density of single- (a) and double-synapse spines (b): sER-free, containing sER, and containing spine apparatus (SA). The graphs show means ± SEM (one-way ANOVA with post hoc Tukey's test, ^∗∗∗ P < 0.001).

Figure 3

PSD area of excitatory ((a) and (b)) and inhibitory (c) synapses of single- and double-synapse spines: sER-free, containing sER, and containing SA. The graphs show means ± SEM (two-way ANOVA with post hoc Bonferroni test; ^∗∗∗ P < 0.001, ^∗∗ P < 0.01, and ^∗ P < 0.05).

Figure 4

Volume of spine apparatus in single- (a) and double-synapse spines (b). The graphs show means ± SEM (one-way ANOVA with post hoc Tukey's test).

Figure 5

Electron micrographs showing polyribosomes in sER-free, sER-containing (a), and SA-containing (b) spines. Arrows: polyribosomes; arrowhead: sER (a) and spine apparatus (b).

Figure 6

Number of polyribosomes in the single- (a) and double-synapse spines (b): sER-free, containing sER, and containing SA. The graphs show means ± SEM (chi square test and one-way ANOVA with post hoc Tukey's test: ^∗∗∗ P < 0.001, ^∗∗ P < 0.01, and ^∗ P < 0.05).

Figure 7

3D reconstruction of single- and double-synapse spines from serial micrographs showing different shapes of spines: stubby (a), thin (b), mushroom (c), and intermediate (d). Color areas as in Figure 1.

Figure 8

Shapes of single- (a) and double-synapse spines (b): thin, mushroom, and stubby. The graphs show percentages of spine types and their numbers inside the bars (chi square test: ^∗ P < 0.05).