Age-related synapse loss in hippocampal CA3 is not reversed by caloric restriction

Abstract

Caloric restriction (CR) is a reduction of total caloric intake without a decrease in micronutrients or a disproportionate reduction of any one dietary component. While CR attenuates age-related cognitive deficits in tasks of hippocampal-dependent memory, the cellular mechanisms by which CR improves this cognitive decline are poorly understood. Previously, we have reported age-related decreases in key synaptic proteins in the CA3 region of the hippocampus that are stabilized by lifelong CR. In the present study, we examined possible age-related changes in the functional microcircuitry of the synapses in the stratum lacunosum-molecular (SL-M) of the CA3 region of the hippocampus, and whether lifelong CR might prevent these age-related alterations. We used serial electron microscopy to reconstruct and classify SL-M synapses and their postsynaptic spines. We analyzed synapse number and size as well as spine surface area and volume in young (10 months) and old (29 months) ad libitum fed rats and in old rats that were calorically restricted from 4 months of age. We limited our analysis to SL-M because previous work demonstrated age-related decreases in synaptophysin confined to this specific layer and region of the hippocampus. The results revealed an age-related decrease in macular axo-spinous synapses that was not reversed by CR that occurred in the absence of changes in the size of synapses or spines. Thus, the benefits of CR for CA3 function and synaptic plasticity may involve other biological effects including the stabilization of synaptic proteins levels in the face of age-related synapse loss.

Figure 1. Hippocampal regions and the location of stratum lacunosum-moleculare (SL-M) within CA3

(A) Low power photomicrograph of a coronal section through the dorsal hippocampus illustrating the dentate gyrus (DG), CA3, and CA1 regions in the Fischer 344XBrown Norway rat. (B) High power photomicrograph of a coronal section depicting the layers of hippocampal area CA3. Quantification of synapses and neurons was confined to SL-M. SL-M, stratum lacunosum-moleculare; SR, stratum radiatum; SL, stratum lucidum. * indicates pyramidal cell layer. Scale bars: (A) 300 μm and (B) 150 μm.

Figure 2. Serial reconstruction of a spine in CA3 SL-M

The series of 12 electron photomicrographs shown in A–L represent a portion of a typical serial series used to reconstruct the synaptic microcircuitry in SL-M. The asterisks in B–J indicate a spine that was reconstructed in this series; four different views of that reconstructed spine and its postsynaptic density (red) are shown at the bottom of the figure. Scale bar: 0.5 μm.

Figure 3. Axo-spinous and axo-dendritic synapses in CA3 SL-M of young AL, old AL, and old CR rats

Electron micrographs illustrating tissue ultrastructure and representative examples of axo-spinous and axo-dendritic synapses in the SL-M of young AL, old AL and old CR rats. Both types of synapses were observed in all groups and no gross abnormalities were observed. Y-AL, young ad libitum fed; O-AL, old ad libitum fed; O-CR, old caloric restricted. Red arrows indicate synapses. Scale bar: 0.5 μm.

Figure 4. Serial reconstructions of postsynaptic densities (PSD) and spines in CA3 SL-M of young AL, old AL, and old CR rats

Based on the PSD morphology, synapses were classified as macular or complex. Macular synapses demonstrated a simple, disk-shaped form; complex synapses demonstrated either partial or complete discontinuities (see Methods). Both types of synapses [macular (A, C, E) and complex (B, D, F)] and a similar range of spine morphologies were found in the all groups. Y-AL, young ad libitum fed; O-AL, old ad libitum fed; O-CR, old caloric restricted. Scale bar: 0.5 μm.

Figure 5. Quantification of macular and complex synapses terminating on spines and dendrites in CA3 SL-M of young AL, old AL, and old CR rats

The total number synapses of each type in the reconstructed volume of SL-M was normalized to the stereologically determined number of neurons in that tissue block. An ANOVA revealed a main effect on macular axo-spinous synapses (A), but not complex axo-spinous synapses (B). Post hoc analysis indicated that the number of macular axo-spinous synapses decreased with age (p<0.05; * indicates significant decrease from young-AL group), but was not reversed by CR. No significant differences were observed for macular (C) or complex (D) axo-dendritic synapses. Data are presented as mean ± standard error of the mean (n = 5). Correlational analysis revealed a significant negative correlation between macular axo-spinous and axo-dendritic synapses (r = −0.51, p = 0.051; E), indicating that axo-dendritic synapses increased as axo-spinous synapses decreased. No relationship was observed for the complex axo-spinous and axo-dendritic synapses (F). Young-AL, young ad libitum fed rats (open bars); Old-AL, old ad libitum fed rats (black bars); Old-CR, old caloric restricted rats (gray bars). Axo-sp: Axo-spinous, Axo-den: Axo-dendritic.