Neurogenesis-independent mechanisms of MRI-detectable hippocampal volume increase following electroconvulsive stimulation

doi:10.21203/rs.3.rs-3215270/v1

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Neurogenesis-independent mechanisms of MRI-detectable hippocampal volume increase following electroconvulsive stimulation

https://doi.org/10.21203/rs.3.rs-3215270/v1

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published 09 Jan, 2024

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Electroconvulsive therapy (ECT) is the most effective psychiatric treatment but the underlying mechanisms are still unclear. In vivo human magnetic resonance imaging (MRI) studies have consistently reported ECT-induced transient hippocampal volume increases, and an animal model of ECT (electroconvulsive stimulation: ECS) was shown to increase neurogenesis. However, a causal relationship between neurogenesis and MRI-detectable hippocampal volume increases following ECT has not been verified. In this study, we were randomly allocated into four groups, each undergoing a different number of ECS sessions (e.g., 0, 3, 6, 9). T2-weighted image was acquired from those mice using 11.7-tesla MRI. A whole brain voxel-based morphometry analysis was conducted to identify any ECS-induced brain volume changes. Additionally, histological examinations with super-resolution microscopy were conducted to investigate microstructural changes in brain regions which show volume changes following ECS. Furthermore, parallel experiments were performed on X-ray irradiated mice to investigate a causal relationship between neurogenesis and ECS-related volume changes. As a result, we revealed for the first time that ECS induced MRI-detectable dose-dependent hippocampal volume increase in mice. Furthermore, ECS increased hippocampal volumes even in mice lacking neurogenesis, suggesting that neurogenesis is not required for the increase. Comprehensive histological analyses identified an increase in excitatory synaptic density in the ventral CA1 as a major contributor to the observed hippocampal volume increase following ECS. Our findings demonstrate that modification of synaptic structures rather than neurogenesis may be the underlying biological mechanism of ECT/ECS-induced hippocampal volume increase.

Biological sciences/Neuroscience/Cellular neuroscience

Health sciences/Anatomy/Nervous system/Brain

electroconvulsive stimulation

neurogenesis

hippocampus

magnetic resonance imaging

synapse

Neuroplasticity refers to the brain’s ability to reorganize neural networks, which can be observed at molecular and cellular levels up through large-scale brain networks. Both human and animal studies have suggested that neuroplastic change in the hippocampus is a shared mechanism for several psychiatric treatments[1]. Among specific treatments for psychiatric disorders, electroconvulsive therapy (ECT) is the most effective. For instance, ECT is effective for pharmacotherapy-resistant depression[2], bipolar disorder[3, 4], and schizophrenia[5]. Thus, a better understanding of the mechanisms underlying ECT efficacy may provide some direction towards the development of new treatments for severe pharmacotherapy-resistant psychiatric disorders.

In vivo human neuroimaging studies have consistently reported hippocampal volume increases following ECT[6, 7], which is consistent with the neuroplastic hypothesis for the mechanisms of ECT[8]. Importantly, the observed hippocampal volume increases could not be explained only by edematous change[9–11]. Whilst the relationship between the hippocampal volume changes and antidepressant effects remains under debate[7, 12, 13], several ECT parameters, such as the number of sessions[7, 12], the effect of electrical stimulation[13], and induced seizure[14], were associated with hippocampal volume increase following ECT as detected by magnetic resonance imaging (MRI). This volume increase is transient[15, 16], and might be reflective of a short window for structural and/or functional network remodeling[17]. Consistent with this idea, a hippocampal subfield analyses revealed that the largest effect of ECT on the hippocampus was observed in the dentate gyrus[16, 18, 19], where neurogenesis in known to occur.

Electroconvulsive stimulation (ECS), an animal model of ECT, was shown to manifest an increased number of newborn cells in the subgranular zone of the dentate gyrus in rodents[20, 21], and nonhuman primates[22]. Additionally, ECS-induced antidepressant-like behavioral changes were not observed in a pharmacogenetic rodent model lacking adult neurogenesis[23], suggesting that ECS requires hippocampal neurogenesis to exert an antidepressant-like effect. Similarly, some behavioral effects of antidepressant medications (e.g., fluoxetine) were blocked when neurogenesis was inhibited[24, 25], and increasing hippocampal neurogenesis may even be sufficient to exert antidepressant- and anxiolytic-like effects[26]. Considering all the evidence from human neuroimaging and preclinical histological studies, one may speculate that the MRI-detectable hippocampal volume increases following ECT could be induced by neuroplastic cellular changes, particularly neurogenesis. However, the results from previous preclinical histological studies cannot be directly translated or associated with the results of human MRI studies, because no previous investigations have provided direct evidence of a causal relationship between neurogenesis and MRI-detectable hippocampal volume increases following ECT.

Here, we applied a reverse-translational approach to investigate the role of neurogenesis in the context of hippocampal volume increases measured by MRI. Our research questions were as follows: 1) Is neurogenesis required for MRI-detectable hippocampal volume increases following ECS, and 2) If neurogenesis is not required for volumetric changes, which microstructural changes may contribute to the MRI-detectable changes in volume? In this study, we first confirmed with MRI that ECS resulted in increased hippocampal volumes. Then, we repeated the same experiment using X-ray-irradiated mice, where neurogenesis was ablated, to show a causal relationship between neurogenesis and MRI-detectable hippocampal volume increases following ECS. Following these experiments, we conducted a comprehensive histological analysis to detect the microstructural changes, which were associated with the hippocampal volume increases.

Detailed explanations of methods are provided in supplemental methods.

Animals

All animal procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Research Committee of Keio University School of Medicine (21031). Experiments were performed using 8-week-old male C57BL/6j mice, which were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan).

ECS

Following anesthesia via 3.0% sevoflurane, ECS was administered to mice once daily, three times a week. To investigate the effect of the number of ECS sessions on brain volumetric changes, mice were allocated to four groups based on the number of ECS sessions (i.e., 0, 3, 6, and 9 sessions). ECS was given via bilateral ear clip electrodes with 25 mA for 1 second at a frequency of 100 Hz square wave pulses (UgoBasile, Comerio, Italy). The sham-treated group was exposed to the same procedure on the same schedule without the electrical stimulation.

X-ray irradiation

The lead plate was placed above the mice to protect their head and body from radiation exposure. The slit in the lead plate was placed just above the whole hippocampus so that other brain regions were not exposed to the radiation. A total dose of 10 Gy was administered to the hippocampus.

MRI analysis

An ex vivo MRI was performed using a 11.7 T BioSpec 117/11 US/R unit (Biospin GmbH, Ettlingen, Germany). One day after the last ECS session, mice were deeply anesthetized and perfused with a 4% paraformaldehyde phosphate buffer solution. The brains were removed along with the skull. For brain volumetric analysis, the structural images were acquired using T2-weighted multi-slice rapid acquisition with relaxation enhancement (RARE). Image preprocessing and statistics for the whole-brain voxel-based analysis were performed using SPM12 (Wellcome Trust Centre for Neuroimaging, London, UK). The segmented images were spatially normalized into the study-specific template. Whole-brain voxel-wise comparisons of gray matter volume (GMV) between the groups were performed using SPM12 with the TBV as a covariate. The first analysis compared the GMV between the ECS-treated mice (ECS) and sham-treated mice (CTL). A second analysis compared the GMV between the X-ray-irradiated mice treated with ECS (X-ray + ECS) and the X-ray-irradiated mice treated with sham ECS (X-ray). The significance threshold for the voxel-wise whole-brain analyses was set at a cluster-level family-wise error (FWE) corrected p < 0.05, with an individual voxel threshold of p = 0.001.

Histology

Our histological analysis investigated following micro-structures with antibodies: neurogenesis (anti-DCX), excitatory terminal (anti-VGluT1), excitatory spine (anti-PSD95), inhibitory terminal (anti-VGAT), PV interneuron (anti-PV), myelin (anti-PLP), neuronal soma (anti-NeuN), astrocyte (anti-GLT1), and microglia (anti-Iba1). These micro-structural changes were analyzed using super-resolution microscopy.

ECS induces MRI-detectable hippocampal volume increases.

Eight-week-old male C57BL/6j mice were randomly allocated into four groups (n = 12 in each group), including sham-ECS (i.e., 0×ECS), 3×ECS, 6×ECS, and 9×ECS, to investigate 1) if 9×ECS induced MRI-detectable changes in brain volumes (i.e., a group comparison between sham-ECS and 9×ECS), and 2) whether the ECS-induced brain volume changes were dose-dependent (i.e., a regression analysis using data from the four groups) (Fig. 1A). An ex vivo MRI assessment was conducted after the last ECS session. Our ECS protocol (i.e., 25 mA for 1 seconds at a frequency of 100 Hz square wave pulses) successfully induced generalized seizures, which were confirmed by electroencephalography (EEG) (Fig. 1B).

Whole-brain voxel-wise analysis of MRI data revealed that ECS increased bilateral ventral hippocampal volumes, particularly in the CA1 and dentate gyrus, whereas ECS did not decrease brain volumes (Fig. 1C and Supplemental Fig. 1). Because in vivo human neuroimaging studies have reported a positive correlation between the number of ECT sessions and the hippocampal volume increase[7], we also investigated the effect of the number of ECS sessions on MRI-detectable hippocampal volume changes. A whole brain regression analysis, including whole brain gray matter volume (GMV) as a dependent variable, the number of ECS sessions as an independent variable, and the total brain volume as a nuisance covariate, revealed that bilateral hippocampal volumes showed significant positive correlations with the number of ECS sessions (left hippocampus: Pearson’s r = 0.71, df = 46, p < 0.001; right hippocampus: Pearson’s r = 0.63, df = 46, p < 0.001) (Fig. 1D, 1E). These results are consistent with the findings from human ECT-MRI studies, suggesting that the results from this ECS-MRI study could be comparable to the results from human studies.

Neurogenesis is not required for the ECS-induced hippocampal volume increases.

A histological analysis confirmed that the ECS protocol significantly increased the number of doublecortin-positive (DCX⁺: a marker of newborn neurons) cells in the dorsal and ventral dentate gyrus (Fig. 2A–C). To investigate the causal relationship between ECS-induced neurogenesis and MRI-detectable hippocampal volume increases (Fig. 1C, 1D), we conducted the same whole-brain analysis using X-ray-irradiated mice (n = 24; 12 received 9×ECS and 12 did not). We confirmed that X-ray irradiation markedly decreased the number of DCX⁺ cells in the dentate gyrus, and that the 9×ECS sessions did not increase the number of DCX⁺ cells in the dentate gyrus (Fig. 2D–F). These results indicate that the X-ray irradiation protocol successfully ablated neurogenesis in the dentate gyrus. Contrary to our expectations, the whole brain MRI analysis revealed that ECS increased bilateral hippocampal volumes, including in the dentate gyrus and CA1, even in the X-ray-irradiated mice (Fig. 2G). The identified brain regions in the analysis were highly overlapped in mice, both with and without X-ray-irradiation.

Overall, our results indicate that 1) ECS induces MRI-detectable hippocampal volume increases in mice, which is consistent with findings from human MRI studies, and 2) ECS-induced increased neurogenesis is not required for ECS-induced, MRI-detectable hippocampal volume changes.

RNAseq screening indicates ECS-induced microstructural changes.

To explore the cellular mechanisms of the MRI-detectable, ECS-induced hippocampal volume increases, we first conducted RNA sequencing (RNAseq) of the 9×ECS-treated hippocampal tissues (Fig. 3A). Based on the results of the whole-brain MRI analysis (Fig. 1C), we focused on the ventral hippocampus. A total of 300 mRNAs were upregulated and 473 were down regulated (Fig. 3B). Using these differential gene expressions (DGEs), we conducted a gene ontology (GO) analysis to examine the associated characteristics. The top significant GO terms from the analysis of the upregulated genes included biological processes related to metabolism, cytoskeleton organization, and cell migration (Fig. 3C). Furthermore, within those involving cellular structure, the top significant GO terms of the upregulated genes involved cellular components related to neuronal microstructure, including the myelin sheath, mitochondria, neuronal cell body, synapses, neuron projections, dendrites, and microtubules (Fig. 3D). In contrast, there were no significant GO terms associated with the down regulated genes. We also investigated changes in the expression of each gene in the hippocampus with ECS (Supplemental Fig. 2). We found that the expression levels of axon-related (e.g., Tubb3 and Nefm), dendrite-related (e.g., Map2), and synapse-related (e.g., Sv2a, Syn1, Syn2, and Nlgn2) genes were increased, indicating ECS-induced, neuronal microstructural changes. In addition, an investigation of the immediate early genes (e.g., fos, Arc, Npas4, and Erg1) showed a reduction in their expressions following ECS, suggesting that 9×ECS might decrease neuronal activity in the hippocampus. In accordance with these results from the RNAseq screening, further histological analyses focused on neuronal microstructural components including axons, dendrites, synapses, and myelin.

ECS increases dendritic branching in the ventral CA1.

We focused on the ventral CA1 (vCA1) in the following histological analyses. As a supplementary analysis, we also investigated the histological changes in the vCA2, but the effects of ECS were mainly observed in the vCA1 (Supplemental Fig. 3). First, we investigated the macrostructural changes by measuring the length of each layer in the vCA1 and dorsal CA1 (dCA1). The length of each layer [stratum oriens (SO) + stratum pyramidale (SP), stratum radiatum (SR), and stratum lacunosum moleculare (SLM)] was defined by the contrast of the vesicular glutamate transporter 1 (VGluT1) immunostaining (Fig. 4A). The analysis indicated that ECS lengthened the SR layer in the vCA1 but not in the dCA1 (Fig. 4B, C). Second, we examined changes in dendritic length and branching by Golgi staining because the SR layer is composed mainly of dendrites from the CA1 neurons. We found that ECS increased the total length of the apical dendrites in the vCA1, as well as the number of dendritic branching points (Fig. 4D–F).

ECS increases excitatory and inhibitory terminals and myelin.

We examined the effect of ECS on microstructural components, including the excitatory and inhibitory terminals, spines, and myelin, using super-resolution microscopy (SRM). First, we found that ECS increased the density, but not the size, of the VGluT1⁺ excitatory terminals in the SR layer of the vCA1 (Fig. 5A–C). We also found that ECS increased synaptic density [i.e., the number of pairs of VGluT1⁺ puncta and post synaptic density 95 (PSD95)⁺ puncta] as well as PSD95⁺ spine sizes in the CA1 (Fig. 5D–F). Golgi staining confirmed that ECS increased the spine density and spine head diameter in the SR of the vCA1 neurons (Supplemental Fig. 4). These results indicate that ECS increased synaptic density in the SR of the vCA1. To investigate the relationship between excitatory synapses and brain volumes measured by MRI, we further conducted correlation analyses using MRI and histological measurements from the same animals. Voxel values in the identified clusters from the VBM analysis were normalized to the total brain volume. Then, the bilateral normalized voxel values were averaged for the subsequent analyses, because the histological analyses did not reveal any indication of laterality. The voxel values in the vCA1 were correlated significantly with the density of the VGluT1⁺ terminals (Pearson’s r = 0.67, df = 34, p < 0.001, R² = 0.44), but not with the VGluT1⁺ terminal size (r=–0.06, df = 34, p = 0.73, R² = 0.004) (Fig. 5G).

Second, we found that the density and the size of the vesicular GABA transporter (VGAT)⁺ inhibitory terminals increased in the SR layer of the vCA1 (Supplemental Fig. 5A–C). The voxel values in the vCA1 were significantly correlated with the VGAT⁺ terminal density (r = 0.55, df = 34, p < 0.001, R² = 0.30), and the VGAT⁺ terminal size (r = 0.39, df = 34, p = 0.02, R² = 0.15) (Supplemental Fig. 5D).

Finally, we found that ECS increased the percent area of myelin proteolipid protein (PLP)⁺ in the SR layer of the vCA1 (Supplemental Fig. 6A, B). The diameter of the myelinated axons increased following ECS, yet myelin thickness did not change (Supplemental Fig. 6C, D). Since myelinated axons in the SR mainly originate from inhibitory interneurons including parvalbumin (PV)⁺ interneurons[27], we examined whether myelin in the SR was increased in PV⁺ interneurons. Although ECS did not increase the PV⁺ axon density, it did increase the density of the PV⁺ myelinated axons (i.e., PLP and PV double positive axons), and the ratio of the PV⁺ myelinated axons to all PV⁺ axons (Supplemental Fig. 6E-H), indicating that PV⁺ interneurons are one type of axons with increased myelin following ECS. The voxel values in the vCA1 were significantly correlated with the percent area of the PLP⁺ myelin (r = 0.47, df = 34, p = 0.004, R² = 0.22) (Supplemental Fig. 6I).

Overall, we found that ECS increased the density of excitatory synapses (both spines and terminals), the density and size of the inhibitory terminals, and the percentage of myelin in the SR layer of the vCA1. Among these, the density of the VGluT1⁺ terminals had the highest correlation with the voxel values in the vCA1 (standardized beta coefficient = 0.55, p = 0.002), suggesting that among the observed microstructural changes, ECS-induced increases in excitatory terminal densities may represent the greatest contribution to the ECS-induced, MRI-detectable hippocampal volume increases. In addition, the number of ECS sessions were positively associated with the density of the excitatory VGluT1⁺ terminals (Pearson’s r = 0.85, df = 34, p < 0.001), inhibitory VGAT⁺ terminals (Pearson’s r = 0.65, df = 34, p < 0.001), and percent area of myelin (Pearson’s r = 0.72, df = 34, p < 0.001) (Supplemental Fig. 7). These results indicate that ECS increased the excitatory and inhibitory synapses as well as myelin in the SR layer of the vCA1 in a dose-dependent manner, similar to the MRI-detectable, dose-dependent hippocampal volume increases.

ECS increases the density and morphology of microglia but not other glial cells.

We examined the effect of ECS on the cellular density and morphology of the vCA1 pyramidal neurons and glial cells. We found that ECS did not change the density of the soma nor the size of the vCA1 pyramidal neurons (Supplemental Fig. 8A, B). In addition, ECS did not change the density of the Gja1⁺ astrocytes nor that of the Plp1⁺ oligodendrocytes. In contrast, ECS increased the density of Csf1r⁺ microglia in the SR layer of the vCA1 (Supplemental Fig. 8C-H). Additionally, ECS increased the percent area of the ionized calcium-binding adaptor molecule 1 (Iba1)⁺ microglia, yet it did not increase the astrocytic glutamate transporter 1 (GLT1)⁺ astrocytes in the SR layer of the vCA1 (Supplemental Fig. 9). These results indicate that ECS increased the volume of activated microglia in the SR.

ECS induces dendritic arborization, and increases the number of excitatory terminals and myelin in mice lacking neurogenesis.

Since our MRI analysis identified hippocampal volume increases following ECS even in X-ray-irradiated mice lacking neurogenesis (Fig. 2G), we also conducted histological analyses of the vCA1 in these mice. First, we found that ECS increased the length of the layer of the SR, the total length of the apical dendrites, and the number of dendritic branches in the vCA1 (Supplemental Fig. 10A–E). Second, we found significant increases in the density of the VGluT1⁺ terminals (t = 2.85, df = 10, p = 0.02) and the percent area of the PLP⁺ myelin (t = 9.36, df = 10, p < 0.01), but not in the density of the VGAT⁺ terminals (t = 1.32, df = 10, p = 0.22) (Supplemental Fig. 10F-H). Golgi staining confirmed that ECS increased spine density and spine head diameter in the SR of the vCA1 (Supplemental Fig. 11). These results indicate that ECS induces dendritic and synaptic changes regardless of the presence of neurogenesis.

In addition, X-ray irradiation itself increased the percent area of the GLT1⁺ astrocytes and Iba1⁺ microglia. However, ECS did not induce additional changes in astrocytes or microglia in the X-ray-irradiated mice (Supplemental Fig. 12). These results suggest that activated astrocytes and microglia do not contribute to the ECS-induced volume increases in the X-ray-irradiated mice.

In the current study, we showed for the first time, that an animal model of ECT induced MRI-detectable hippocampal volume increases in a dose-dependent manner using an unbiased, whole brain, voxelwise analysis. This result is consistent with findings from human neuroimaging studies[6, 7]. Because previous human studies have reported the effect of ECT was greatest in the dentate gyrus where neurogenesis occurs[10, 16, 19], and ECS strongly enhances adult neurogenesis in the dentate gyrus of rodents, we hypothesized that neurogenesis was necessary for the MRI-detectable volume increases. Contrary to our expectations, however, the MRI-detectable hippocampal volume increases following ECS were observed even in mice lacking neurogenesis, suggesting that neurogenesis is not required for the ECS-induced volume increases. Our comprehensive histological analyses indicated that dendritic arborization, synaptic structural modifications, and increased myelin might contribute to the MRI-detectable, dose-dependent hippocampal volume increases following ECS. Our results provide the first evidence of the cellular mechanisms underlying ECS-induced, MRI-detectable, hippocampal volume increase, which may be considered a reasonable interpretation of the findings from human ECT-MRI studies.

In vivo human MRI-measurements (e.g., gray matter volumes) are a valuable tool to noninvasively assess brain structural plasticity. However, combined MRI and histological studies are required to directly link MRI-derived measures with underlying cellular alterations, because MRI-derived measures alone are not specific to particular underlying microstructural components[28]. An important and unexpected finding in the present study is that the MRI-detectable hippocampal volume increases following ECS were observed regardless of the presence of neurogenesis. Histological analysis revealed increased excitatory and inhibitory terminals, as well as myelin in the CA1, which correlated with MRI-measurements (Fig. 5 and Supplemental Fig. 5–7). Among those microstructural changes, alterations in the density of the excitatory terminals showed the highest correlation with the hippocampal volume increases following ECS. These results suggest that changes in the excitatory terminals were the major contributor to the hippocampal volume increases following ECS.

In addition to the changes in terminals, ECS also increased the number of dendritic branches and spine density, both of which were reported as the basis for MRI-detectable GMV changes[29, 30], and were observed in previous ECS studies[31, 32]. Although it is difficult to identify the cellular component which contributed the most to the volume increase, our results suggest that a modification of synaptic structures (e.g., dendritic spines and axon terminals) induces the ECS-induced hippocampal volume increases in the vCA1. In line with our findings, a previous study reported ECS-induced increases in the number of spines in the CA1[33]. Because the majority of excitatory synapses are generally formed on dendritic spines, the previous result may suggest that the main effect of ECS was on excitatory synapses. In addition, an increase in the number of spines in the CA1 has also been reported as an effect of antidepressant medications[34, 35], suggesting a shared mechanism for different antidepressant treatments. Morphological changes in dendrites and synapses could alter neurotransmission, synaptic function, and neural plasticity[36]. Hence, future studies should investigate how the histological changes in the CA1 might be related to changes in hippocampal function and antidepressant-like effects. It should be noted that this result did not indicate that the vCA1 was the primary target region of ECS because the VGluT1⁺ excitatory terminals in the SR layer of the vCA1 were mainly projections from the dorsal and ventral CA3[37]. ECS may primarily induce plastic changes in the CA3 pyramidal neurons or neurons in the dentate gyrus, which are more upstream subnuclei within the hippocampal circuitry.

Although ECS also increased inhibitory terminals, our post-hoc additional analyses suggested a possibility that the VGAT⁺ inhibitory terminals did not increase linearly in a dose-dependent manner, but rather they decreased with less than three ECS sessions and increased with more than six ECS sessions (Supplemental Fig. 7B, C). Our results from additional histological analyses suggest that a small number of ECS sessions enhanced the excitatory input to the vCA1, whereas repeated ECS sessions also enhanced inhibitory input to the vCA1. Each stage of ECS could exhibit differential effects on excitatory and inhibitory input. Future studies should investigate the early and late effects of ECS on the excitatory-inhibitory balance during a course of ECS. Our results from the RNAseq analysis, which showed decreased neural activity markers following 9×ECS sessions, support decreased neural activity in the late stages of ECS.

Our histological analysis revealed for the first time that ECS increased myelin in the vCA1. In addition, the neuronal subtype exhibiting increased myelin was the PV⁺ inhibitory interneuron. Some studies have reported that chronic stress reduces PV⁺ inhibitory interneurons in the hippocampus, particularly in the CA1, CA2/3, and dentate gyrus, and that antidepressant medications also increased the number of the PV⁺ neurons. However, these findings have been inconsistent[38]. In addition, human postmortem studies reported decreased PV⁺ interneurons in patients with mood disorders[39, 40]. Although it is speculative, our findings of the ECS-induced increases in myelin in the vCA1, might be associated with the underlying antidepressant mechanisms of ECS. However, there is a possibility that ECS also increased other myelinated axons, including other subtypes of interneurons, which we did not investigate because it was well beyond the aims of the present study.

Some limitations of this study should be acknowledged. First, we conducted an ex vivo VBM analyses, but not an in vivo VBM assessment, in this study to detect the hippocampal volume change. Compared to in vivo VBM analyses, the fixation procedure consisting of paraformaldehyde (PFA) and phosphate buffer solution (PBS) strongly influences volume changes[41]. The volume changes depend on the duration, for which the brain was immersed in PFA for post-fixation and in PBS for storage. In this study, the duration of PFA and PBS immersion was strictly controlled for all samples to minimize the duration-dependent differences in ex vivo brain volume. Ex vivo VBM analysis can obtain high quality data with high space resolution, high signal-noise ratio, and high contrast, as compared to in vivo. Second, this study was conducted using wild-type mice. Thus, we did not investigate whether the MRI-detectable hippocampal volume changes and the observed changes in the neurogenesis-independent, hippocampal volume increases following ECS were correlated with antidepressant-like effects. However, our research questions in this study were to investigate if ECS would induce MRI-detectable hippocampal volume changes, and whether neurogenesis was necessary for this change. Because we confirmed in this study that ECS induced MRI-detectable brain volume increases in mice that were similar to human studies, a future study should include a mouse model of depression to explore how the MRI / histological changes might be associated with the potent antidepressant-like effect of ECS. Furthermore, the developed experimental and analysis pipeline can be used to probe the cellular mechanisms of other treatment interventions and may provide microstructural evidence for results from human MRI studies.

Data availability

All data in this study are available from the corresponding author upon request. The RNAseq dataset is provided in the Supplementary Data in this paper.

Acknowledgement

We thank Dr. Toru Suzuki (Keio University School of Medicine) for his technical assistance with the histological studies. We thank the Collaborative Research Resources, Keio University School of Medicine, for the technical assistance with the Zeiss ELYRA 3D-SIM system.

Funding

This work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) KAKENHI Grain Number JP21K20911/22K15756 (A.T.), JP22K15215 (Y.A.), Takeda Science Foundation (Y.A.), Astellas Foundation for Research on Metabolic Disorders (Y.A.), a grant from Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) by the Japan Agency for Medical Research and Development (AMED) under grant numbers JP19dm0207069 (K.F.T) and JP21wm0525018 (S.Y.).

Author Contributions

A.T. supervised the project, Y.A, K.F.T, and A.T. designed the experiments, Y.A, S.Y., and A.T. performed and analyzed the MRI studies, Y.A, K.Y., and A.T. conducted ECS on the animals, Y.A. performed all histological experiments, T.K. performed the EEG experiments, and Y.A. and A.T. wrote and revised the original manuscript.

Conflict of interest

The authors declare no competing interests.

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Neurogenesis-independent mechanisms of MRI-detectable hippocampal volume increase following electroconvulsive stimulation

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