Aging-independent decrease of complex multi-spine boutons in hippocampal area CA1 after contextual fear conditioning

Sep 30, 2026

Long-lasting synaptic changes support memory storage and regulate recall. Prior work showed that multi-innervated dendritic spines (MISs) drive hippocampal memory formation in aged, but not young, mice after contextual fear conditioning. That study used ultrastructural analysis in CA1 24 h after training, without accounting for multi-spine boutons (MSBs). Re-analysis of 3D electron microscopy images reveals an unexpected reduction of complex MSB complexity in CA1 stratum radiatum 24 h after conditioning. This decrease occurs in both young and aged mice, indicating aging does not affect the synaptic change. Complex MSBs couple multiple postsynaptic neurons, so a decline in complexity may be required for specific memory recall.

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Abstract

Introduction

Figure 1: MSB complexity after contextual fear conditioning

Martinez-Serra et al. Molecular Brain (2026) 19:1 [https://doi.org/10.1186/s13041-025-01265-z](https://doi.org/10.1186/s13041-025-01265-z) Molecular Brain MICRO REPORT Open Access Aging-independent decrease of complex multi-spine boutons in hippocampal area CA1 after contextual fear conditioning Raquel Martinez-Serra 1, Suji Lee 1, Igor Kraev2 and Karl Peter Giese 1* Abstract Long-lasting synaptic changes enable memory storage and regulate recall in the brain. Our previous work established that generation of multi-innervated dendritic spines (MISs), spines with typically two excitatory presynaptic inputs, underlies hippocampal memory formation in aged, but not young mice. The identification of MIS generation was done by ultrastructural analysis in hippocampal CA1 stratum radiatum 24 h after contextual fear conditioning (CFC) . However, our analysis did not consider multi-spine boutons (MSBs), which were recently shown to increase in complexity (complex MSBs are pre-synaptic boutons connecting with more than two postsynapses) at a later time point after CFC in young age. Therefore, we re-analyzed our three-dimensional electron microscopy images and show that, unexpectedly, MSB complexity, decreases in CA1 stratum radiatum 24 h after CFC. The decrease in MSB complexity occurred both in young and aged mice, indicating that aging has no impact on this synaptic change. Considering that complex MSBs link the activity of multiple postsynaptic neurons, we suggest that after CFC a decrease in MSB complexity may be required for specific memory recall. Keywords Multi-synapse bouton, Hippocampus, Memory, Aging Introduction The hippocampus has a critical role for memory [1], and with aging hippocampal memory formation declines [2]. However, hippocampal memory can still be formed and stored in old age. Using experimental conditions under which young and aged mice form equally a hippocampus-dependent contextual fear memory, we found evidence that the synaptic basis of memory changes with aging [3]. Specifically, we found that (structural) longterm potentiation (LTP) in hippocampal CA1 stratum *Correspondence: Karl Peter Giese [karl.giese@kcl.ac.uk](karl.giese@kcl.ac.uk) 1Department of Basic and Clinical Neuroscience, King’s College London, London, UK 2Electron Microscopy Facility, Faculty of Science, Technology, Engineering and Mathematics, Open University, Milton Keynes, UK radiatum underlies contextual memory formation in young mice, but not in aged mice. Instead, generation of multi-innervated spines (MISs), which involves attraction of a presynaptic input onto an existing dendritic spine [4], underlies contextual memory formation in aged, but not young mice. This finding not only illustrates that the synaptic basis of memory changes with age, it also points toward an importance of specific types of synaptogenesis that lead to generation of multi-synapses. Most synapses exist in a 1–1 ratio between pre-synaptic and post-synaptic terminals. However, multisynapses, connections between more than two synaptic profiles, have been observed in significant abundance in mammalian brains of several species, including humans [5]. The most abundant multi-synapses are multi-spine boutons (MSBs), where one pre-synaptic bouton connects to multiple spines. Our previous analysis [3] did © The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use

FAQs

What change in hippocampal synapses occurs 24 hours after contextual fear conditioning?

Complex multi-spine bouton (MSB) complexity decreases in CA1 stratum radiatum 24 h after contextual fear conditioning.

Does aging affect the decrease in MSB complexity after contextual fear conditioning?

No. The reduction occurs in both young and aged mice, indicating aging has no impact on this synaptic change.

Why are complex multi-spine boutons important for memory recall?

Because complex MSBs connect multiple postsynaptic neurons, the study suggests that decreasing complex MSB complexity may be required for specific memory recall after conditioning.

Sep 30, 2026

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