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Characterization of Multiple Synaptic Boutons in Cerebral Motor Cortex in Physiological and Pathological Condition: Acrobatic Motor Training Model and Traumatic Brain Injury Model
Applied Microscopy 2018;48:102-9
Published online December 28, 2018
© 2018 Korean Society of Microscopy.

Hyun-Wook Kim1, Ji eun Na1, and ImJoo Rhyu1,2,*

1Department of Anatomy, Korea University College of Medicine, Seoul 02841, Korea, 2Division of Brain Korea 21 Plus Program for Biomedical Science, Korea University College of Medicine, Seoul 02841, Korea
Correspondence to: *Correspondence to: Rhyu IJ,, Tel: +82-2-2286-1149, Fax: +82-2-929-5696, E-mail:
Received December 19, 2018; Revised December 23, 2018; Accepted December 23, 2018.
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Multiple synaptic boutons (MSBs) have been reported to be synapse with two or more postsynaptic terminals in one presynaptic terminal. These MSBs are known to be increased by various brain stimuli. In the motor cortex, increased number of MSB was observed in both acrobat training (AC) model and traumatic brain injury (TBI) model. Interestingly one is a physiological stimuli and the other is pathological insult. The purpose of this study is to compare the connectivity of MSBs between AC model and TBI model in the cerebral motor cortex, based on the hypothesis that the connectivity of MSBs might be different according to the models. The motor cortex was dissected from perfused brain of each experimental animal, the samples were prepared for routine transmission electron microscopy. The 60~70 serial sections were mounted on the one-hole grid and MSB was analyzed. The 3-dimensional analysis revealed that 94% of MSBs found in AC model synapse two postsynaptic spines from same dendrite. But, 28% MSBs from TBI models synapse two postsynaptic spines from different dendrite. This imply that the MSBs observed in motor cortex of AC model and TBI model might have different circuits for the processing the information.

Keywords : Multiple synaptic boutons (MSBs), Synaptic plasticity, Acrobat training (AC), Traumatic brain injury (TBI)
Fig. 1. Photograph of acrobat training task and mean time for AC model and error rate. (A) AC model traversing a rod, ladder, chain bridge, walls, double rod, and grid floor during 4 trials per day, for 20 days. (B) Mean time for complete a trial was significantly decreased as AC model progressed (Pearson correlation coefficient; r=−0.95; p<0.0001; left). Mean number of errors per trial were also decreased during 20 days training (Pearson correlation coefficient; r=−0.5763, p=0.0039; right).
Fig. 2. Photograph of traumatic brain injury model. (A) The anesthetized mice were fixed on stereotaxic to induce TBI, and then placed in a liquid nitrogen-cooled probe for 1 minute in the exposed brain region. (B) The photograph of cardiac perfused mouse brain at 5day after TBI.
Fig. 3. Illustration for two different models of multiple synaptic boutons depend on connection. MSBs could be divided into two models, one is spine pairs arose from same dendrite (sdMSBs, left) might be enhance local synaptic efficacy, the other is spines origination from different dendrite (ddMSBs, right) suggests reorganization of neural networking. S, spine.
Fig. 4. Synapse distribution of cerebral motor cortex in AC model. Effects of IC and AC on the number of SSBs and MSBs. (A) Total number of synapses include SSBs with MSBs were significantly increased after AC in cerebral motor cortex layer V (p=0.0016; Student t-test). (B) However, effective changes number of synapses occur in MSBs (SSBs, p=0.4699; MSBs; p<0.0001; Student t-test). (C) Estimation for number of synapses per unit volume (1 μm3) presented same results comparison total number of synapses significantly (SSBs, p=0.4698; MSBs, p<0.0001; Student t-test). (D) Proportion of SSBs and MSBs were showed similar result between IC and AC. Data are means±SEM.
Fig. 5. Synapse distribution of cerebral motor cortex in traumatic brain injury model. Synapse distribution of WT and TBI mice in cerebral motor cortex layer V. (A) Total number of synapses includes SSBs and MSBs were changed just in after TBI 15 day model (TBI_5D, p=0.0533; TBI_15D, p=0.0330; TBI_30D, p=0.2244; Student t-test). (B) However, estimated number of MSBs were significantly increased in all TBI models (TBI_5D, p=0.0019; TBI_15D, p<0.0001; TBI_30D, p=0.0003; Student t-test). (C) Also, number of MSBs in per unit volume (1 μm3) was significantly different in WT mice and TBI models (TBI_5D, p=0.0019; TBI_15D, p<0.0001; TBI_30D, p=0.0003; Student t-test). (D) Proportion of SSBs and MSBs were showed similar result between WT mice and TBI model analyzed per unit volume. Data are means ± SEM.
Fig. 6. Spine pairs origin of cerebral motor cortex in AC model and TBI model. The results of three dimensional tracing of MSBs for spine pair origin confirmation both two groups using Reconstruct® software. Proportion of sdMSBs was significantly increased in AC model (sdMSBs, p=0.0017; Student t-test). However, ddMSBs were major connection type of MSBs in TBI after 15 day model (ddMSBs, p=0.0135; Student t-test).
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