The effects of aging on neuropil structure in mouse somatosensory cortex-A 3D electron microscopy analysis of layer 1

Abstract

This study has used dense reconstructions from serial EM images to compare the neuropil ultrastructure and connectivity of aged and adult mice. The analysis used models of axons, dendrites, and their synaptic connections, reconstructed from volumes of neuropil imaged in layer 1 of the somatosensory cortex. This shows the changes to neuropil structure that accompany a general loss of synapses in a well-defined brain region. The loss of excitatory synapses was balanced by an increase in their size such that the total amount of synaptic surface, per unit length of axon, and per unit volume of neuropil, stayed the same. There was also a greater reduction of inhibitory synapses than excitatory, particularly those found on dendritic spines, resulting in an increase in the excitatory/inhibitory balance. The close correlations, that exist in young and adult neurons, between spine volume, bouton volume, synaptic size, and docked vesicle numbers are all preserved during aging. These comparisons display features that indicate a reduced plasticity of cortical circuits, with fewer, more transient, connections, but nevertheless an enhancement of the remaining connectivity that compensates for a generalized synapse loss.

Fig 1. Somatosensory cortex of aged mice is thinner than adults.

A, Semi-thin coronal sections of mouse somatosensory cortex indicating a reduced cortical thickness in the aged example (left, adult, 4 months old; right, aged, 24 months old). B, Measurements of cortical thickness from 3 aged and 3 adult mice, in the semi-thin sections, showed a reduction of 15.6%, (adult, 1.03 ± 0.047 mm; aged, 0.87 ± 0.003 mm; unpaired t-test, p = 0.0297). C, Measurements of layer I thickness from the same mice, in the semi-thin sections, showed a reduction of 19.4% (adult, 0.103 ± 0.004 mm; aged, 0.083 ± 0.002 mm; N = 10 per each of the six mice; p = 0.0009, unpaired t-test). D, Counts of cell profiles from sections used in B, across the cortical thickness, show no difference between adult and aged animals in any of the five bins positioned from the pial surface to the white matter. Bars indicate the mean ± sem. Differences are not statistically significant; Kolmogorov-Smirnov test, p > 0.9. Scale bar in A is 100 micrometers.

Fig 2. Synapse density decreases per unit volume in adult and aged mouse layer 1 neuropil.

A, To count synapses in stacks of serial FIBSEM images (3 adult, and 3 aged), circles were placed at the center of each synapse using the TrakEM2 software in FIJI (www.fiji.sc); with their diameter equal to the maximum diameter of each contact. B, micrographs showing: left, an asymmetric synapse on a spine (presumed glutamatergic, black arrowhead) and a symmetric synapse on a shaft (presumed inhibitory, white arrowhead); center, an asymmetric synapse on a shaft; right, a multi-synaptic bouton (MSB). C, The density of all synapses was significantly lower in aged mice (adult, 1.40 ± 0.006 per μm³; aged, 1.17 ± 0.013 per μm³; t-test, *, p < 0.001) with a significant drop in asymmetric synapses on dendritic spines (adult, 1.05 ± 0.03 per μm³; aged, 0.96 ± 0.02 per μm³; t-test, *, p < 0.001). On dendritic shafts, there were less asymmetric synapses, but the drop was not significant (adult, 0.17 ± 0.03 per μm³; aged, 0.11 ± 0.02 perμm³; t-test, p = 0.09). D, Symmetric (presumed inhibitory) synapses were also significantly reduced, but only on dendritic spines (adult, 0.07 ± 0.01 per μm³; aged, 0.02 ± 0.006 per μm³; t-test, *, p = 0.0028) and not on shafts (adult, 0.08 ± 0.07 per μm³; aged, 0.065 ± 0.003 per μm³; t-test, p = 0.27). Scale bar in B is 500 nanometers.

Fig 3. Synapses are larger in aged layer 1 neuropil.

A, FIBSEM electron micrograph shows two boutons making asymmetric synapses; synapse 1 is made with a single synaptic bouton, 2 and 3 with a multi-synaptic bouton. B, the same synapses were segmented in the TrakEM2 software in FIJI (shown in red: www.fiji.sc) and reconstructed in 3D. C, There is a strong correlation between the diameter of circles used to annotate 57 synapses and their surface areas measured from their 3D reconstruction (slope of second polynomial regression, y = 0.032–0.09x + 0.95x²; R² = 0.84). D, Frequency distribution of asymmetric synapse sizes, estimated from the maximum diameter measurements shown in Fig 2 (Inset shows average values of all measurements, N = 2800 adult synapses, 377.8 ± 3.2 nm; N = 2423 aged synapses, 427.7 ± 3.9 nm; p < 0.0001, Kolmogorov-Smirnov test), showed that synapses in layer 1 of aged mice are larger. E, The same data plotted on a logarithmic scale for adult, and F, aged mice. Both show a log-normal distribution (adult, red fitting; center = 287.5, amplitude = 7.27, width = 0.33, R² = 0.96; aged, blue fitting; center = 305.1, amplitude = 4.9, width = 0.47, R² = 0.94). G, Examples of a series of micrographs containing a perforated synaptic density (top) and its segmentation (bottom) by means of the automated detection method, and its 3D rendering on the right. H, Graph showing the percentage of asymmetric synapses that displayed one or more perforation.

Fig 4. Dense reconstructions of sub-volumes reveal the same amount of asymmetric synaptic surface area per unit length of axon and dendrite, and per unit volume of neuropil in adult and aged mice.

A, FIBSEM images were segmented in the Ilastik software (www.ilastik.org) to reconstruct all the axons (shown in blue) and dendrites (in green). Synapses were segmented in the TrakEM2 software in FIJI (shown in red: www.fiji.sc). B, These stacks were sub-volumes from the larger ones used for synapse density measurements (Fig 2A) and had side lengths of 5μm (volumes of 25 μm³). All axons, dendrites and synapses were reconstructed in six of these cubes (3 adults, 3 aged). C, Average synaptic surface area shows a significant increase in aged animals (adult, N = 313 synapses 0.15 ± 0.007 μm² per μm³; aged, N = 288 synapses 0.17 ± 0.008 μm² per μm³; Kolmogorov-Smirnov test, p = 0.04). D, Analysis of neurite lengths shows that layer 1 neuropil of aged mice contained significantly more micrometers of axon per cubic micrometer of neuropil (8.15 ± 0.19 μm of axons per μm³ for adults; 10.27 ± 0.88 μm of axons per μm³ for aged; p = 0.016, unpaired t-test). The dendritic content was smaller but non-significant (1.37 ± 0.06 μm of dendrites per μm³ for adults; 1.53 ± 0.12 μm of dendrites per μm³ for aged; p = 0.3, unpaired t-test). E, Number of asymmetric synapses per unit length of axon was 32.9% less in aged mice (adult, 0.173 ± 0.005 synapses per μm of axons; aged, 0.116 ± 0.011 synapses per μm of axons; t-test, p = 0.008). Numbers of asymmetric synapses per unit length of dendrite was 24.6% less in aged mice (adult, 1.03 ± 0.046 synapses per μm of dendrite; aged, 0.776 ± 0.072 synapses per μm of dendrite; t-test, p = 0.04). F, Asymmetric synaptic surface area per unit length of axons or dendrites was not significantly different mice (adult, 0.065 ± 0.012 μm² per μm of axons; aged, 0.055 ± 0.007 μm² per μm of axons; unpaired t-test, p = 0.53; adult, 0.144 ± 0.026 μm² per μm of dendrites; aged, 0.126 ± 0.014 μm² per μm of dendrites; unpaired t-test, p = 0.57). G, Asymmetric synaptic surface per unit volume also showed no difference between the adult and aged mice (adult, 0.19 ± 0.03 μm² per μm³; aged, 0.19 ± 0.012 μm² per μm³; t-test, p = 0.9).

Fig 5. Spine and bouton volume correlate closely with synapse size in the aged and adult neuropil.

A, Histogram showing the distribution of vesicle density normalized to the synaptic contact surface area shows no difference between aged and adult animals (p = 0.4, Kolmogorov-Smirnov test). B, Correlation between spine head volume and synaptic surface area for adult (N = 207; R² = 0.673) and aged (N = 197; R² = 0.817) mice. The slopes are not significantly different (ANOVA, p = 0.42). C, Correlation between volume of excitatory boutons and synaptic surface area for adult (N = 169; R² = 0.369) and aged (N = 146; R² = 0.526) mice. The slopes are significantly different (ANOVA, p = 0.0045).

Fig 6. Different compartments unchanged in aged neuropil.

A, The volumes of every mitochondria were measured from reconstructions in the sub-volumes. B, The volume fraction of mitochondria in axons, dendrites and glia did not change between the two groups (total; adults 9.8 ± 0.76%, aged 9.5 ± 0.37%, p = 0.76; axons, adults 3.7 ± 0.15%, aged 3.9 ± 0.45%, p = 0.71; dendrites, adults 5.1 ± 0.7%, aged 5.1 ± 0.4%, p = 0.94; others, adults 1.1 ± 0.25%, aged 0.6 ± 0.26%, p = 0.22; unpaired t-test). C, The distribution of mitochondrial sizes is not different between the adult and aged mice. Frequency distribution histogram shows the densities of different mitochondria, grouped by volume (adults 0.116 ± 0.06 mitochondria per μm³, aged 0.127 ± 0.07 mitochondria per μm³, p = 0.99; Kolmogorov-Smirnov test). D, Percentage of occupancy of connecting units (boutons and spines combined), axons, dendrites, glia, as well as mitochondria, per cubic micrometer of neuropil from the each of 6 sub-volumes. None of the differences are statistically significant (Connecting unit, adults 29.4 ± 1.96%, aged 29.4 ± 1.96%, p = 0.4; axons; adults, 19.9 ± 1.49%, aged 24.1 ± 1.46%, p = 0.11; dendrites, adults 26.7 ± 1.09%, aged 28.9 ± 2.81%, p = 0.52; glia, adults 23.9 ± 1.1%, aged 20.8 ± 1.73%, p = 0.21; mitochondria, adults 9.8 ± 0.76%, aged 9.5 ± 0.37%, p = 0.76; N = 3 per each group, unpaired t-test).