Rapid and stable changes in maturation-related phenotypes of the adult hippocampal neurons by electroconvulsive treatment
© The Author(s). 2017
Received: 11 August 2016
Accepted: 22 February 2017
Published: 2 March 2017
Electroconvulsive therapy (ECT) is a highly effective and fast-acting treatment for depression. Despite a long history of clinical use, its mechanism of action remains poorly understood. Recently, a novel cellular mechanism of antidepressant action has been proposed: the phenotype of mature brain neurons is transformed to immature-like one by antidepressant drug treatments. We show here that electroconvulsive stimulation (ECS), an animal model of ECT, causes profound changes in maturation-related phenotypes of neurons in the hippocampal dentate gyrus of adult mice. Single ECS immediately reduced expression of mature neuronal markers in almost entire population of dentate granule cells. After ECS treatments, granule cells showed some of physiological properties characteristic of immature granule cells such as higher somatic intrinsic excitability and smaller frequency facilitation at the detate-to-CA3 synapse. The rapid downregulation of maturation markers was suppressed by antagonizing glutamate NMDA receptors, but not by perturbing the serotonergic system. While single ECS caused short-lasting effects, repeated ECS induced stable changes in the maturation-related phenotypes lasting more than 2 weeks along with enhancement of synaptic excitation of granule cells. Augmentation of synaptic inhibition or blockade of NMDA receptors after repeated ECS facilitated regaining the initial mature phenotype, suggesting a role for endogenous neuronal excitation in maintaining the altered maturation-related phenotype probably via NMDA receptor activation. These results suggest that brief neuronal activation by ECS induces “dematuration” of the mature granule cells and that enhanced endogenous excitability is likely to support maintenance of such a demature state. The global increase in neuronal excitability accompanying this process may be relevant to the high efficacy of ECT.
KeywordsAntidepressant Electroconvulsive seizure Hippocampus Maturation Granule cell
Granule cells (GCs) in the hippocampal dentate gyrus (DG) have been implicated in the pathophysiological mechanisms of neuropsychiatric disorders including depression and schizophrenia, and have been suggested to be an important target for both pharmacological and physical therapeutic treatments [1–6]. We have recently demonstrated distinct changes in the molecular and physiological phenotypes of GCs as a candidate cellular mechanism of action of a selective serotonin reuptake inhibitor (SSRI): The SSRI fluoxetine can transform several mature features of GCs in the adult mouse DG . The fluoxetine treatment strongly reduced expression of mature GC markers, induced active somatic membrane properties resembling immature GCs, and decreased short-term plasticity at the dentate-to-CA3 synaptic connection that characterizes the mature dentate-to-CA3 signal transmission. These changes cannot be explained simply by an increase in newly generated immature GCs, but are most likely characterized as “dematuration” of mature GCs .
These changes in the maturation-related phenotypes of GCs were not observed after short-term treatment of fluoxetine, and the efficacy of fluoxetine is quite variable among individual mice [7, 8]. Such unstable and unpredictable nature of the effects of fluoxetine well mimics common clinical observations in antidepressant medication such as delayed emergence of therapeutic effects and treatment resistance . In a mouse model of depression and anxiety, the changes in the maturation-related phenotypes of GCs can be induced at a serum concentration of fluoxetine close to that seen in patients during fluoxetine medication , suggesting clinical relevance of these changes in GCs. Therefore, we proposed that modifications of maturation-related phenotypes in mature neurons may cause some beneficial or therapeutic effects of SSRIs by reinstating cellular functions of immature neurons. This proposal has been supported by demonstration of similar SSRI-induced changes in the neuronal maturation status in other brain regions [11, 12].
In addition to antidepressant drugs, electroconvulsive therapy (ECT) has been used to treat depression. ECT was originally devised for treating psychosis in the 1930s  and is currently considered as the most effective treatment for depression . ECT has fast-acting antidepressant effects and is effective in most of medication-resistant patients. Despite a long history of clinical use, the mechanism of action of ECT still remains poorly understood. In particular, it is unknown how distinct types of treatments, antidepressant drug medication and ECT, converge on the same antidepressant action. In rodents, ECT-like stimulation has been shown to reduce immunoreactivity for the calcium binding protein calbindin in the DG [15, 16]. Calbindin has been considered as a marker for mature GCs, because its expression is not seen in newly generated GCs at the cell age of 10 days or less and is established in about 4 weeks . A reduction of calbindin expression is one of characteristic features of demature GCs in SSRI-treated mice. Therefore, it is possible that ECT-like stimulation can also induce dematuration of GCs, possibly in a more rapid and consistent manner than SSRI treatments. To test this hypothesis, in the present study, we examined how electroconvulsive stimulation (ECS), an animal model of ECT, regulates maturation-related phenotypes of the mature GCs in the adult mouse DG.
Downregulation of mature granule cell markers by ECS
ECS-treated granule cells partially exhibit physiological properties resembling immature neurons
ECS-treated mice share gene expression changes in DG with SSRI-treated mice and genetically modified mice with altered GC maturation
Functional classification on the basis of the gene ontology (GO) terms
Increased change by both ECS- and SSRI-treatment (660 probes)
cholesterol metabolic process
sterol biosynthetic process
steroid metabolic process
cholesterol biosynthetic process
steroid biosynthetic process
nervous system development
negative regulation of neuron projection development
positive regulation of neuron projection development
response to drug
response to cAMP
response to stress
cellular response to extracellular stimulus
response to cytokine
negative regulation of signal transduction
lipid metabolic process
positive regulation of apoptotic process
response to estrogen
transforming growth factor beta receptor signaling pathway
negative regulation of MAP kinase activity
negative regulation of extrinsic apoptotic signaling pathway
positive regulation of gene expression
Functional classification on the basis of the gene ontology (GO) terms
Decreased change by both ECS- and SSRI-treatment (670 probes)
nervous system development
positive regulation of transcription from RNA polymerase II promoter
transmembrane receptor protein tyrosine kinase signaling pathway
interleukin-1-mediated signaling pathway
positive regulation of cell proliferation
cellular response to calcium ion
Most up-regulated genes by ECS treatment among genes significantly regulated by both ECS- and SSRI-treatment (Top 15 genes)
αCaMKII hetero KO
Most down-regulated genes by ECS treatment among genes significantly regulated by both ECS- and SSRI-treatment (Top 15 genes)
αCaMKII hetero KO
Involvement of NMDA receptor-dependent signaling in the rapid downregulation of maturation markers by ECS
We then examined the involvement of an N-methyl-D-aspartate receptor (NMDAR) using D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDAR antagonist. Intracerebroventricularly injected D-AP5 attenuated the reduction of Calb1 and Tdo2 expression levels by a single ECS (Fig. 5c). As NMDAR activation upregulates expression of many genes in the DG, gene induction and subsequent de novo protein synthesis may be necessary for ECS-induced downregulation of mature GC markers. As expected, pretreatments with cycloheximide, a protein synthesis inhibitor, attenuated the reduction of Calb1 and Tdo2 by a single ECS (Fig. 5d). These results suggested that protein synthesis and NMDAR activation are important for the rapid downregulation of mature GC markers by ECS.
Long-lasting phenotypic change in granule cells after repeated ECS
Role of GABAergic signaling in reversal of long-lasting phenotypic change
Increased synaptic activation of granule cells after repeated ECS
This continuous enhancement of synaptic activation in GCs through, and also beyond, the period of ECS treatments suggests a possible involvement of endogenous neuronal excitation during the non-stimulated period in the establishment of the lasting change in the maturation-related phenotype of GCs. To test this possibility, we examined the effect of an NMDAR antagonist applied during the inter-stimulus period. The NMDAR antagonist, 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), was administered during the latter half of ECS treatments. To minimize the effect of CPP on NMDAR activation that was directly induced by ECS, CPP was injected shortly after each ECS. This CPP treatment significantly reversed the reduction of frequency facilitation at 14 days after 11 times of ECS (Fig. 8f). Even a single CPP injection after the last ECS was effective in reversing the reduction of frequency facilitation (Fig. 8f). These results suggest that the endogenous neuronal activity contributes to the lasting change in the maturation-related phenotype of GCs via NMDAR activation.
We have previously shown that the SSRI antidepressant fluoxetine can induce GC dematuration [7, 8, 10, 20]. The phenotype of ECS-treated GCs investigated here resembled those of the SSRI-treated demature GCs, as exemplified by similar comprehensive gene expression profiles in the DG. Since ECS has been considered to be an animal model of the ECT used for depression, our results suggest that dematuration of neurons in the DG could be a common cellular basis for pharmacological and physical antidepressant treatments. One of the common functional features of the GCs found in SSRI- and ECS-treated mice is increased somatic excitability. In addition, ECS-treated GCs show alteration of the synaptic E/I balance toward excitation. It has been suggested that neuronal excitation or the E/I balance in the DG is perturbed by chronic stress [27–29]. It is possible that GC dematuration restores neuronal activity in the DG of individuals in a depressed state by enhancing the synaptic and/or intrinsic excitability. Clinically, ECT has fast-acting antidepressant effects and is effective in most of patients who are resistant to monoaminergic antidepressant drugs. Consistent with this clinical setting, the phenotypic change in GCs by ECS emerges rapidly and does not require the serotonergic system. Although the magnitude of dematuration, as assessed by the reduction of frequency facilitation, is on average similar between ECS and SSRI, the effect of ECS is much less variable than that of SSRIs (compare Fig. 3g in this study and Fig. 3b in ). In SSRI-treated mice, the excessive change in the maturation-related phenotype was accompanied by a marked increase in day-to-day fluctuation of home-cage activity levels that resembled an antidepressant-induced rapid change in mood . In addition, α-CaMKII hetero KO mice show profound immaturity of the DG and infradian oscillation of home-cage activity levels . Thus, the invariable effect of ECS may be related to a relatively low risk for switching to mania in patients with bipolar disorder who are treated with ECT . While ECT rapidly alleviates acute episodes of depression, relapse rates are relatively high after discontinuation of the therapy [31, 32]. Based on our model, increased neuronal activity and/or augmentation of NMDAR activation may help to prevent relapse of depressive symptoms after discontinuation of ECT.
We demonstrated that the synaptic E/I balance is changed toward excitation in the ECS-treated GCs, and that the excitability of the ECS-treated GCs is continuously enhanced. Enhanced GABAergic inhibition by diazepam after the period of ECS treatments partially reversed the altered phenotype of GCs to the initial matured state (Fig. 7e), suggesting that endogenous neuronal activity is required for lasting maintenance of the demature state of GCs. Since block of NMDAR activation during the non-stimulated period also attenuated the long-lasting effect of ECS on the phenotype of GCs, it is likely that the enhanced synaptic excitation of these cells supports their demature state via NMDAR activation. Recent studies including our own have suggested that both changes in the E/I balance and aberrant neuronal maturation are involved in the pathophysiological basis of schizophrenia and related neuropsychiatric disorders [2, 5, 26, 33–36]. It is possible that defects in neuronal maturation could alter the circuit E/I balance, and a change in the E/I balance toward over-excitation may anchor the aberrant state of maturation in these disorders. Enhanced inhibition may benefit this type of psychiatric disorder, not only by correcting the circuit E/I balance, but also by allowing proper maturation of neurons in the circuit. Indeed, enhancing GABAergic inhibition has been suggested as a treatment strategy for schizophrenia . Further investigation of the regulation of neuronal maturation by neuronal activity would facilitate understanding of the heterogeneous pathogenic and pathophysiological mechanisms underlying neurodevelopmental psychiatric disorders.
Male C57BL/6J or C57BL/6N mice, 8 weeks of age, were purchased from Japan SLC or Charles River Japan. The 5-HT4 receptor mutant mice (strain name: B6.129P2-Htr4 < tm1Dgen>/J > backcrossed to the C57BL/6J background more than 10 times were purchased from the Jackson Laboratory. Male and female homozygous mutant mice and their wild-type littermates from heterozygous mating were used in this study. Mice were pair-housed and maintained under standard conditions with a 12-h light/dark cycle and ad libitum access to food and water unless otherwise stated. All mice were habituated for over one week before experimental procedures.
Bilateral ECS (current, 25 mA; shock duration, 1 s; frequency, 100 pulse/s; pulse width, 0.5 msec) was administered via moistened, spring-loaded ear-clip electrodes with a pulse generator (ECT Unit; Ugo Basile) to mice that were anesthetized with isoflurane (1.5 to 2%) in order to avoid sudden unexpected death associated with seizures. In repeated treatments, ECS was administered 4 times a week for up to 3 weeks. The sham-treated animals were handled in an identical manner to the ECS-treated animals without the administration of shock. In cranial irradiation experiments, ECS was started 14 day after irradiation. Cranial X-ray irradiation was administered as previously described . X-ray (Rigaku Radiofrex 350 X-ray generator) at a dose of 10 Gy was delivered at a dose rate of 0.74 Gy/min.
Fluoxetine hydrochloride (LKT Labs) was dissolved in drinking water and orally applied at a dose of 22 mg/kg/day for 4 weeks as described previously . The fluoxetine solutions were prepared every day, and daily fluoxetine intake was determined for individual mice on the basis of the water consumption during the preceding 24 h and the body weight measured every other day. Saccharin (0.2%) was included in the fluoxetine solution to keep water intake comparable to the baseline. Diazepam (Wako) was dissolved in dimethyl sulfoxide (DMSO) at 10 to 20 mg/ml, diluted in the drinking water, and administered at 5 to 10 mg/kg/day. D-AP5 (Tocris Bioscience) was dissolved in saline and intracerebroventricularly administered (1 μg/mouse) under anesthesia with pentobarbital (50 mg/kg) 20 min prior to ECS. Cycloheximide (Santa Cruz) was dissolved in saline and intraperitoneally administered at a dose of 200 mg/kg 30 min before ECS. For deletion of serotonergic neurons, 5,7-DHT (Sigma Aldrich) was dissolved in saline containing 0.1% ascorbic acid and was intracerebroventricularly administered (200 μg/mouse) under anesthesia with pentobarbital (50 mg/kg) 30 min after intraperitoneal injection of desipramine (25 mg/kg; Sigma Aldrich). The deletion of serotonergic neurons 1 week after the injection was confirmed in equally conditioned mice by immunohistochemical analysis using anti 5-HT antibody (Immunostar 20080, diluted 1:10000). CPP (Sigma-Aldrich) was dissolved in saline and intraperitoneally injected at a dose of 20 mg/kg shortly after ECS procedures.
Real time PCR
Primer sequences used for real-time RT-PCR
Mice were perfused with saline and 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were dissected out and postfixed in the same fixative at 4 °C for 24 h. After immersion in 0.1 M phosphate buffer containing 20% sucrose at 4 °C overnight, the brains were rapidly frozen at -80 °C and sectioned using a cryostat at 30 μm thickness. The free-floating sections were first incubated with 10% normal equine serum in PBS containing 0.3% Triton X-100 for 1 h at room temperature and subsequently incubated with mouse anti-calbindin-D-28 K monoclonal antibody (Sigma Aldrich, C9848, diluted 1:3000), mouse anti-calretinin monoclonal antibody (Millipore, MAB 1568, diluted 1:3000), goat anti-doublecortin polyclonal antibody (Santa Cruz, sc-8066, diluted 1:500), rabbit anti-c-Fos polyclonal antibody (Calbiochem, PC38, diluted 1:2000), or mouse anti-NeuN antibody (Millipore, MAB377, diluted 1:500) overnight at 4 °C. After washing three times with PBS containing 0.3% Triton X-100, the sections were incubated with secondary antibody conjugated with AlexaFluor488 or AlexaFluor555 (Molecular Probes). After washing, the sections were mounted on slides and observed with a fluorescent microscope (Biozero 8000, Keyence). For some sections of doublecortin immunostaining, biotinylated horse anti-goat IgG (Vector) was used as a secondary antibody, followed by incubation with ABC Vectastain Kit (Vector). Antigen detection was performed with 3,3′ -diaminobenzidine staining.
Mice were singly housed in the institutional standard condition (14:10 light/dark cycle; lights on at 6:00 A.M. through 8:00 P.M.). Mice were decapitated under deep halothane anesthesia 24 h after the last ECS unless otherwise stated. Both hippocampi were isolated, and transverse hippocampal slices (380 μm) were cut using a tissue slicer. Electrophysiological recordings were performed as described . Recordings were made in a submersion-type chamber maintained at 27.0–27.5 °C and superfused at 2 ml/min with saline composed of (in mM): NaCl, 125; KCl, 2.5; NaH2PO4, 1.0; NaHCO3, 26.2; glucose, 11; CaCl2, 2.5; MgCl2, 1.3 (equilibrated with 95% O2/5% CO2). EPSPs arising from the MF synapses were evoked by stimulating the dentate granule cell layer and recorded from the stratum lucidum of CA3 using a glass pipette filled with 2 M NaCl. The amplitude of field EPSPs was measured on analysis as described . A criterion used to identify the MF input was more than 85% block of EPSP by an agonist of group II metabotropic glutamate receptors, (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV, 1 μM). Single electrical stimulation was delivered at a frequency of 0.05 Hz unless otherwise specified. For recording EPSPs and population spikes evoked by activation of the MPP input to GCs, the recording electrode was placed in the granule cell layer, and electrical stimulation was delivered every 30 s via the electrode placed in the middle third of the molecular layer. The initial slope of EPSPs was measured on analysis. The amplitude of the population spike was measured as the difference between the negative peak and the average of two positive peaks. Whole-cell current-clamp recordings were made from GCs with a pipette filled with a solution composed of (in mM) potassium gluconate 140, HEPES 20, NaCl 8, MgATP 2, Na2GTP 0.3, EGTA 0.05 (pH adjusted to 7.3 with KOH). The recording pipette was placed in the middle third of the granule cell layer. Hyperpolarizing currents (6 - 16 pA, 400 ms) were injected through the recording pipette to measure the input resistance. Depolarizing currents (400 ms) with increasing intensity by 10 pA steps were injected to measure the threshold current intensity to evoke action potentials. Whole-cell voltage-clamp recordings were made from GCs with a pipette filled with a solution composed of (in mM) cesium gluconate 140, HEPES 20, NaCl 8, EGTA 0.1, MgATP 2, Na2GTP 0.3 (pH adjusted to 7.3 with CsOH). EPSCs evoked by MPP stimulation were recorded at the reversal potential (-70 mV) for IPSCs. The amplitude of EPSC was adjusted around 100 pA. Monosynaptic IPSCs were then recorded in the same cells at +1 mV in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM) and D-AP5 (25 μM). The liquid junction potential was corrected in these recordings. All recordings were made using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA), filtered at 2 kHz and stored in a personal computer via an interface (digitized at 5 - 10 kHz). DCG-IV and CNQX were purchased from Tocris Bioscience (Bristol, UK). Picrotoxin was from Wako Pure Chemical Industries, Ltd (Osaka, Japan).
In situ hybridization
In situ hybridization was performed using a digoxigenin (DIG)-labeled riboprobe as described . Calb1 cDNA template probe was cloned by PCR with gene-specific primers (Forward; 5′-actgaccacagcggcttc-3′, Reverse; 5′-agaggcagaagcccatcc-3′, Product Length; 927 bp), verified by sequencing, and used to produce a labeled riboprobe with the RNA Labeling kit (Roche). The brains were removed from mouse skulls at 6 h after single ECS and rapidly frozen on dry ice. None of the sense probes yielded any signal.
For comparison of gene expression profiles between single ECS-treated and repeated ECS-treated DG, the same RNA samples as in Fig. 2a and b were used. Dematured DG and control samples were collected as follows. Mice were decapitated at 24 h after the 11 times of ECS or 4-week treatment of fluoxetine at a dose of 22 mg/kg. The hippocampal slices were prepared as described above. The dentate gyrus was dissected from some of them, and the remaining slices were used for electrophysiological analyses. Frequency facilitation at the MF-CA3 synapse was measured in each mouse, and the DG samples from fluoxetine-treated mice that exhibited small frequency facilitation were used as dematured DG (n = 3). Reduced facilitation was also confirmed in ECS-treated mice. The control DG samples were collected from vehicle- or sham-treated mice. Total RNA was extracted by using an RNeasy micro kit (Qiagen) and the samples of the same groups were put together. From each group, 100 ng of total RNA was amplified with 3′IVT Express kit (Affymetrix, Inc.). All samples were hybridized to the GeneChip mouse genome 430A 2.0 array (Affymetrix, Inc.), and the microarray suite 5.0 of the Affymetrix gene chip operating software was used for the analysis of the GeneChip data. For each transcript represented on the array, the statistical expression algorithm computes detection (present or absent), signal intensity, change (increase or decrease), and change p-value. For the comparison in the DG between repeated ECS- and chronic fluoxetine-treatment, if the average signal intensity in all of the conditions (ECS, sham, fluoxetine, control) was lower than 50, the genes were excluded from further analysis. Hippocampal gene expression data in Shn-2 KO mice and αCaMKII hetero KO mice was obtained from  and , respectively. Correlations were examined with Spearman correlation coefficients. Gene ontology analysis was performed using DAVID Bioinformatics Resources 6.8 (National Institute of Allergy and Infectious Diseases).
The brains were removed at 24 h after the last ECS and the DG of the hippocampus was dissected under a stereoscopic microscope. The isolated DG was sonicated in protein lysis buffer containing protease inhibitor cocktail (Nacalai tesque) on ice and centrifuged at 20,000 g for 10 min at 4 °C. Supernatants containing 10 μg of proteins were separated on 12% SDS-polyacrylamide gel by electrophoresis and transferred onto polyvinyl difluoride membrane. The membrane was first blocked with Blocking One (Nacalai tesque) for 1 h at room temperature and then incubated with mouse anti-calbindin-D-28 K monoclonal antibody (diluted 1:3000) or anti-PSD-95 monoclonal antibody (BD bioscience, BD610496, diluted 1:500) at 4 °C overnight. After washing, the membrane was incubated with horseradish peroxidase conjugated secondary antibody (Jackson) for 1 h at room temperature, and bands were visualized with the ECL reagent (GE Healthcare). The same membrane was then stripped, and detection of β-actin was performed using mouse anti-β-actin monoclonal antibody (Millipore, MAB1501R, diluted 1:3000) in the same way. The signal intensity of calbindin was calculated by LAS-3000 (FujiFilm) and normalized to that of β-actin.
All data are presented as means ± SEM. Experiments with two groups were compared with unpaired two-tailed Student’s t test unless otherwise specified, and experiments with more than two groups were subjected to one-way ANOVA, followed by the Dunnett’s test or the Tukey’s test. Interaction between subgroups was compared with two-way ANOVA, followed by the Bonferroni’s test. Statistical significance was set at P < 0.05. The number of data “n” represents the number of mice used unless otherwise specified.
serotonin type 4 receptor
Excitatory postsynaptic currents
Excitatory postsynaptic potentials
Immediate early genes
Inhibitory postsynaptic currents
Medial perforant path
Selective serotonin reuptake inhibitor
We thank Yasunori Mikahara, Yoko Oda and Tomoko Nishisaka for technical assistance, and Drs. Kazuhisa Nakayama, Yukihiko Sugimoto, Tomoyuki Furuyashiki, Ronald Duman and Mu-ming Poo for helpful discussion.
This work was supported by JSPS KAKENHI Grant Number 25460096 (to E. S-N), 25116525 (to K.K.), and 15H01296 (to K.K.); The Naito Foundation (to E. S-N); the foundation for Pharmaceutical Sciences (to E. S-N); Brain Science Foundation (to K.K.); Takeda Science Foundation (to K.K.) and Japan Science and Technology Agency, Core Research for Evolutional Science and Technology (to K.K and H.S.).
Availability of data and materials
Microarray data were deposited at the GEO server (GSE54307 and GSE93732).
YI, ES-N, and KK designed the project and wrote the manuscript. YI, and KK performed all experiments. HS participated in the design of the study. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Animal use and procedures were in accordance with the National Institute of Health guidelines and approved by the Committee of Animal Research of Kyoto University, Faculty of Pharmaceutical Sciences, the Animal Care and Use Committee of Tokyo University of Science, and the Animal Care and Use Committee of Nippon Medical School.
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