Increased Axonal Bouton Stability during Learning in the Mouse Model of MECP2 Duplication Syndrome

Abstract

MECP2 duplication syndrome is an X-linked form of syndromic autism caused by genomic duplication of the region encoding methyl-CpG-binding protein 2 (MECP2). Mice overexpressing MECP2 demonstrate social impairment, behavioral inflexibility, and altered patterns of learning and memory. Previous work showed abnormally increased stability of dendritic spines formed during motor training in the apical tuft of primary motor cortex (area M1) corticospinal neurons in the MECP2 duplication mouse model. In the current study, we measure the structural plasticity of axonal boutons in layer 5 pyramidal neuron projections to layer 1 of area M1 during motor training. In wild-type littermate control mice, we find that during rotarod training the bouton formation rate changes minimally, if at all, while the bouton elimination rate more than doubles. Notably, the observed upregulation in bouton elimination with training is absent in MECP2 duplication mice. This result provides further evidence of an imbalance between structural stability and plasticity in this form of syndromic autism. Furthermore, the observation that axonal bouton elimination more than doubles with motor training in wild-type animals contrasts with the increase of dendritic spine consolidation observed in corticospinal neurons at the same layer. This dissociation suggests that different area M1 microcircuits may manifest different patterns of structural synaptic plasticity during motor training.

Keywords: MECP2; autism; bouton; motor learning; plasticity; synaptic.

Figure 1.

Bouton classification and density of L5 pyramidal neuron axonal projections to layer 1 of mouse primary motor cortex (M1). A, In vivo 2-photon imaging. (1) A cranial window is drilled centered 1.6 mm lateral to the bregma to expose area M1. Correct localization to the forelimb was confirmed post hoc by electrical microstimulation (for review, see Ash et al., 2017). (2) GFP-labeled pyramidal neuron processes in layer 1 of area M1 are imaged. The yellow box is shown at high magnification in B. B, Top, The 2-photon image of a 0.1-μm-diameter fluorescent bead, revealing the resolving power of the microscope to be 0.4 µm full-width at half-maximum. Bottom, Example small (1.2-µm-diameter) bouton at the same magnification for comparison, showing that the resolution of the microscope allows ready discrimination of the boutons in this study. C, Bouton classification. Left, Varicosities along axons are classified as alpha boutons (greater than ∼2 µm diameter, blue arrows) or beta boutons (1–2 µm diameter, yellow arrows) based on size (see Materials and Methods). Extraneous fluorescence structures masked for illustration purposes only. Right, Histogram of bouton diameters measured in a subset of axons (n = 54 alpha, n = 74 beta boutons), demonstrating a bimodal distribution. D, E, Histogram of densities of alpha (D) and beta (E) boutons per axonal segment in MECP2 duplication mice (orange, n = 54 segments from seven mice) and WT littermates (black, n = 58 segments from six mice). F, The 4 d spontaneous bouton turnover rate (boutons formed + boutons eliminated)/2*axon length, for alpha boutons and beta boutons. Alpha boutons were highly stable in this time frame.

Figure 2.

Bouton elimination increases during motor training in L1 of WT motor cortex. A, Experimental paradigm and imaging time points. Sample images of axonal segments imaged before (left) and after (middle) 4 d of rotarod training to identify axonal bouton formation (green arrow) and elimination (red arrow) during training. Segments are imaged again following 4 d of rest (right) to identify boutons formed, eliminated, and maintained during rest and training-associated boutons that are stabilized (light green) or not stabilized (pink). Extraneous fluorescence structures were masked and image was slightly smoothed for illustration purposes only. B, Bouton formation and elimination during training (black) and during rest (gray). Bouton elimination was significantly elevated during training, p = 0.001, n = 58 segments, Mann–Whitney U test. Total number of boutons studied: 314 baseline, 40 formed during training, 42 formed during rest, 64 eliminated during training, 23 eliminated during rest. Data were acquired from six mice. Statistics were performed across axonal segments. C, Pre-existing bouton survival curves were across imaging days. The dotted line depicts baseline bouton survival and is calculated from the study by Grillo et al. (2013). D, The fraction of boutons maintained during the rest period, measured for pre-existing boutons (present on day 0) that were still present on day 4 following training (black) and boutons formed during training (training-associated boutons, gray). p = 10⁻⁶, Mann–Whitney U test.

Figure 3.

Increased stability of axonal boutons during training in MECP2 duplication mice. A, Bouton formation during training (training-associated boutons) and during rest in MECP2 duplication mice and WT littermates. B, Pre-existing bouton elimination during training and during rest in each genotype. C, Pre-existing bouton survival curves across imaging. D, The training-associated bouton stabilization rate, the number of boutons formed during training and still present after 4 d of post-training rest, is not significantly different across genotypes. Data are plotted as the percentage of boutons formed during training. Statistics: A–C, linear mixed-effects model ANOVA; D, Mann–Whitney U test.

Figure 4.

Sketch of structural plasticity phenotypes in dendrites and axonal projections in area M1 of MECP2 duplication and WT mice. A highly simplified diagram of the layer 1 motor cortex circuit, including major local connections, inputs, and outputs. The imaged input projection is shown on the right in bold and represents axonal projections to L1 from L5 pyramidal neurons in somatosensory, premotor, and contralateral motor cortex. In WT mice (navy blue), spine formation increases in L5B neuron apical dendrites during motor training, while bouton elimination increases in L5 axonal projections. In MECP2 duplication mice (orange), spine formation/stabilization increases even more than in WT mice during training, while bouton elimination is unchanged. See the text for details.