Maternal dietary loads of alpha-tocopherol increase synapse density and glial synaptic coverage in the hippocampus of adult offspring

Abstract

An increased intake of the antioxidant α-Tocopherol (vitamin E) is recommended in complicated pregnancies, to prevent free radical damage to mother and fetus. However, the anti-PKC and antimitotic activity of α-Tocopherol raises concerns about its potential effects on brain development. Recently, we found that maternal dietary loads of α-Tocopherol through pregnancy and lactation cause developmental deficit in hippocampal synaptic plasticity in rat offspring. The defect persisted into adulthood, with behavioral alterations in hippocampus-dependent learning. Here, using the same rat model of maternal supplementation, ultrastructural morphometric studies were carried out to provide mechanistic interpretation to such a functional impairment in adult offspring by the occurrence of long-term changes in density and morphological features of hippocampal synapses. Higher density of axo-spinous synapses was found in CA1 stratum radiatum of α-Tocopherol-exposed rats compared to controls, pointing to a reduced synapse pruning. No morphometric changes were found in synaptic ultrastructural features, i.e., perimeter of axon terminals, length of synaptic specializations, extension of bouton-spine contact. Glia-synapse anatomical relationship was also affected. Heavier astrocytic coverage of synapses was observed in Tocopherol-treated offspring, notably surrounding axon terminals; moreover, the percentage of synapses contacted by astrocytic endfeet at bouton-spine interface (tripartite synapses) was increased. These findings indicate that gestational and neonatal exposure to supranutritional tocopherol intake can result in anatomical changes of offspring hippocampus that last through adulthood. These include a surplus of axo-spinous synapses and an aberrant glia-synapse relationship, which may represent the morphological signature of previously described alterations in synaptic plasticity and hippocampus-dependent learning.

Figure 1.

Morphometric analyses of axo-spinous synapses and glia-synapse relationships. Examples of micrographs from CA1 stratum radiatum used for morphometrical analyses, showing axo-spinous synapses surrounded by distal astrocytic processes (A and C). Schematic drawings reproducing the corresponding micrographs are depicted in B and D. A and B, an axon terminal (axt1) contacted extra-synaptically (i.e. far away from the cleft) by an astrocytic process (asp1), and two dendritic spines (sp1 and sp3) contacted by extra-synaptic glial processes (asp2 and asp3) are shown; asp2, extra-synaptic on sp1, is also in direct apposition with axt2/sp2 bouton-spine interface, thus being intra-synaptic on this synapse. In C and D, a synapse is contacted at its bouton-spine interface by an astrocytic process (intra-synaptic) covering both pre- and post-synaptic profiles. The membrane apposition between neuronal synaptic profiles and glial processes is marked with dotted lines; bouton-spine interfaces (bold black line) and postsynaptic specializations (double bold gray line) are also marked. As an example of morphometric analyses carried out in the study, measures calculated for axt1/sp1 synapse (in B) are as follows: axt1 perimeter, 2396 nm; portion of axt1 profile covered by asp1, 238 nm (glia-covered/total profile ratio: 0.1); sp1 perimeter, 1310 nm; portion of sp1 profile covered by asp2, 332 nm (glia-covered/total profile ratio: 0.25); length of post-synaptic specializations in sp1, 585 nm and 67 nm; length of axt1/sp1 bouton-spine apposition, 800 nm. sp, dendritic spine; axt, axon terminal; asp, astrocytic process. Scale bars: A,C) 250 nm.

Figure 2.

The density of hippocampal synapse is increased in adult offspring from tocopherol-supplemented mothers. Histogram showing the results of quantitative analysis of the density of axo-spinous synapses in CA1 stratum radiatum of adult rats born to mothers fed supranutritional doses of α-T (TREAT) and age-matched controls (CTRL). Density is calculated as number of synapses per µm². Data are presented as mean values ± SEM. Synapse density is significantly higher in adult rats exposed to α-T during development. Student’s t-test; * P<0.05.

Figure 3.

Morphological organization of neuron-glia relationships at hippocampal synapses are affected by maternal loads of α-T. Histograms in A and B report the average ratio between the linear portion of axon terminal (A) or dendritic spine (B) profile covered by glia and their total perimeter length (excluding bouton-spine apposition length). For axon terminals (A), this ratio is significantly higher in adult offspring from tocopherol-supplemented dams (TREAT) respect to controls (CTRL), indicating a more extended glial coverage of presynaptic boutons. In contrast, no differences were found for dendritic spines (B). Histogram in C shows the percentage of axo-spinous synapses contacted by astrocytic endfeet at bouton-spine interface over the total synapses. This percentage is significantly higher in α-T-treated animals (TREAT) respect to controls (CTRL), pointing to a relative surplus of tripartite synapses in CA1 of adult offspring of α-T-supplemented mothers. Data are presented as mean values ± SEM. Student’s t-test; * P<0.05.