Abstract
Antidepressants that target the glutamatergic system, such as ketamine, have shown promising results. However, the contribution of other potential medications targeting the glutamate system to depression is unclear. Our research found that perampanel can exert a rapid and long-lasting antidepressant effect to improve the behavior of chronic social defeat stress (CSDS) depression susceptible mice. In mechanism, perampanel reduce the abnormal increase in the expression of the NMDA receptor subunit GluN2B (Grin2b) in the medial prefrontal cortex (mPFC), further increase the expression of the AMPA receptor subunit GluA1 (Gria1), and improve the functional impairment of excitatory synaptic transmission in the mPFC in depression susceptible mice. Meanwhile, Naïve mice (non-stressed controls) administered perampanel would rapidly exhibit depression-like behaviors. This is because perampanel inhibits the function of excitatory synaptic transmission in the mPFC of Naïve mice. Indeed, perampanel shows different effects in depression susceptible mice and Naïve mice. Our research reveals that an FDA-approved perampanel for clinical treatment of epilepsy, has rapid antidepressant potential. This finding may provide more treatment options and precise medication guidance for clinical patients with depression and for those with epilepsy accompanied by depression.
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Introduction
Major depressive disorder (MDD) is one of the most prevalent mental health disorders worldwide, resulting in a significant disease burden [1, 2]. Most of the antidepressants commonly used in clinical practice are selective serotonin reuptake inhibitors (SSRIs), which tend to be slow-acting and have a low efficacy rate. They are also prone to relapse [3,4,5,6,7]. However, since esketamine was launched in the United States in 2019, numerous clinical studies have confirmed that ketamine has a rapid and effective antidepressant effect in patients with depression. This is undoubtedly a revolutionary development in the treatment of MDD [8,9,10]. However, ketamine still carries the risk of addiction and hallucinations [11,12,13]. The antidepressant effects of ketamine are not entirely dependent on NMDAR antagonism; AMPAR also participates in the antidepressant effects of ketamine, particularly through increased expression of GluA1 in the medial prefrontal cortex (mPFC) [14,15,16,17,18,19]. This suggests that medications targeting the neural glutamate system may have significant potential in antidepressant treatment.
Perampanel is a novel, clinically approved antiepileptic medication and non-competitive antagonist of neuronal AMPA receptors [20,21,22]. Epilepsy with depression occurs in a high percentage of patients in the clinic [23]. Previous studies have shown that, GluA1 protein expression decreased in brain tissue after administration of perampanel to epileptic mice [24]. AMPAR-mediated postsynaptic membrane depolarization is a prerequisite for NMDAR channel opening, which means that AMPAR inhibition can indirectly inhibit NMDAR [17, 25,26,27,28]. However, it remains unclear whether perampanel exerts depression-related effects through its action on the neuronal glutamate system.
Here, we investigated the role of interactions between AMPA receptors and NMDA receptor subunits in modulating the function of neuronal excitatory synaptic transmission in the glutamate system, and their involvement in the antidepressant effects induced by perampanel. Our comprehensive approach combined behavioral assessment, molecular biology and electrophysiological functional analysis with adenovirus-mediated intervention targeting NMDAR subunit expression. Our results show that interactions between glutamate receptors contribute to the antidepressant properties of perampanel. Therefore, our data may provide new insights for the accurate treatment of patients with epilepsy comorbid with depression using perampanel.
Materials and methods
Mice
C57BL/6 J mice or CD1 mice were housed in an EVC cage at 22 ± 1 °C, humidity 40% under standard laboratory conditions with a 12 h light/dark cycle and with free access to food and water. C57BL/6 J mice (aged 8 weeks) were obtained from the Southern Medical University Animal Center (Guangzhou, China). The adult male CD1 mice (CD-1®(ICR) Mice, NO. VM0011) (older than 5 months of age) were purchased from Charles River Laboratories (Beijing). Before the behavioral tests, the C57BL/6 J mice were handled every day for 3 days and double-blind behavioral experiments were performed. All of the experiments were conducted in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (China) and were approved by the Southern Medical University Animal Ethics Committee (NO. 2016104).
Drug injection
Perampanel was dissolved in a vehicle of 1% DMSO in saline and administered via intraperitoneal (i.p.) injection at a dose of 0.5 mg/kg. The injection volume was standardized at 10 mL/kg of body weight. Mice in the control group received an i.p. injection of the vehicle solution only.
Behavioral studies
Chronic social defeat stress
For the Chronic social defeat stress (CSDS) protocol, adult male CD1 mice (>5 months) were purchased from Charles River Laboratories (Beijing). Experimental C57BL/6 J intruder mice were exposed to CD1 aggressor mice for 10 min. After the 10 min social defeat experiment, the CD1 mice and the C57BL/6 J intruder mice were housed in the same cage but separated by partitions for the remainder of the day. The procedure was repeated for 10 consecutive days, using a different invasive CD1 mouse each day.
Social interaction test
Long-term behavioral consequences of the CSDS were tested using the social interaction (SI) 24 h after the CSDS. Social avoidance behavior was tested using a two-stage social interaction test. In all behavioral experiments, animal trajectories were monitored by a video tracking system. Mice were placed in a new cage (44 × 44 × 44 cm3) containing an empty metal cage (9.5 × 9.5 × 8 cm3) and their movements were tracked for 2.5 min in the absence of an aggressor. Their movements were then tracked for 2.5 min in the presence of the aggressor held in the cage. After each trial, the apparatus was cleaned with a 70% aqueous solution of ethanol to remove olfactory cues and all behavioral tests were conducted in the dark. The SI index (time spent in the interaction zone when the target mouse was present versus when the target mouse was not present) was calculated. They were considered susceptible when they scored <1 and resilient when they scored ≥1.
Forced swimming test
The forced swimming test (FST) was performed in a 45 cm high, 19 cm diameter glass cylinder filled to 23 cm with water (23 °C). Mice were placed inside the cylinder. The duration of the test was 6 min. The mice were acclimated for the first 2 min and the resting period was recorded for the last 4 min. The resting state of the mice was recorded using Ethovision XT software (Noldus, USA).
Tail suspension test
In the tail suspension test (TST), the mouse’s tail is taped 1 cm above the tip and then suspended from a horizontal bar, with its head approximately 15 cm above the ground during suspension. A camera is used to record the mice behavioral responses in the suspended state, and the duration of immobility within a 6-minute period is analyzed and recorded.
Open field test
The mice were placed in an open chamber (40 × 40 × 30 cm) made of grey polyvinyl chloride. The mice were allowed to move freely inside the chamber. The mice were gently placed in the centre and allowed to explore the area for 5 minutes. The total distance travelled and the time spent by the mice in the center (20 × 20 cm2) were recorded by the VersaMax animal activity monitoring system and analyzed using the VersaMax 4.20 software.
Conditioned position preference test
Conditioned position preference (CPP) was used in a three-chamber experimental design. The two chambers (20 ∗ 20 ∗ 50 cm) were distinguished by different wall colors (black and striped white) and floor patterns (spoke and grid) and were separated by a corridor. A video tracking system was used to record mouse movements. Mice were handled and acclimated for 3 d. On day 0, mice were allowed to freely explore the apparatus for 15 min and time spent in the chamber was recorded, excluding mice that spent more than 70% of their time on either side. Conditioning was performed on days 1–7 with the black chamber combined with an intraperitoneal injection of 0.5 mg/kg perampanel for 45 min, followed by the white chamber combined with the same amount of saline for 45 min after 4 h. On day 8, mice were allowed to freely explore the entire apparatus for 15 min and the time spent in the chambers was recorded. the CPP ratio was defined as the amount of time spent in the black chamber divided by the amount of time spent in the two dense chambers.
Prepulse inhibition
The prepulse inhibition (PPI) test was performed on mice using the SR-Lab system (San Diego Instruments, USA). The experimental setup consisted of a Plexiglas cylinder, a transducer, an acoustic (loudspeaker), a lamp and a fan. The mouse was immobilised in a small Plexiglas cylinder, which was placed on a piezoelectric transducer that measured and quantified vibrations and fed them into a computer. The background noise was 65 dB and the duration was 5 min. A total of 42 stimuli per experiment were divided into seven different types of stimuli, i.e., no stimuli, purely startle acoustic stimuli (120 dB for 40 ms), and five different pre-inhibitory acoustic stimuli (74/78/82/86/90 dB for 20 ms, and 120 dB stimuli appearing every 100 ms for 40 ms). Each different type of acoustic stimulus randomly formed a stimulus string, for a total of 6 stimulus strings. Sixty-five milliseconds after the appearance of the startle sound, the vibration levels of the mice were recorded. Forty-two of the same six stimuli were averaged to obtain the value of the vibration level for that type of stimulus. The PPI percentage was calculated using the following formula: [1 - (average startle response to pre-pulse before startle stimulus/average response to startle stimulus)] × 100.
Elevated plus maze
The apparatus consisted of two opposing open arms (30 × 5 × 0.5 cm3) and two opposing enclosed arms (30 × 5 × 15 cm3), which were connected by a central platform (5 × 5 cm2) and positioned 50 cm above the ground. The behavior was tracked for 5 min with an overhead camera and EthoVision 11.0 software (Noldus).
Library preparation and RNA sequencing
Total RNA was isolated from the tissues using the Magzol Reagent (Magen, China) according to the manufacturer’s protocol. The quantity and the integrity of RNA yield were assessed by using the K5500 (Beijing Kaiao, China) and the Agilent 2200 TapeStation (Agilent Technologies, USA), respectively. Briefly, the RNA were fragmented to approximately 200 bp. Then, the RNA fragments were subjected to first strand and second strand cDNA synthesis following by adaptor ligation and enrichment with a low-cycle according to instructions of NEBNext® Ultra RNA LibraryPrep Kit for Illumina (NEB, USA). The final library product was assessed with Agilent 2200 TapeStation and Qubit® (Life Technologies, USA) and then sequenced on Illumina (Illumina, USA) platformat with pair-end 150 bp at Ribobio Co. Ltd (Ribobio, China).
Date preprocessing
Adaptor and low-quality bases were trimmed with Trimmomatictools(version:0.36), and the clean reads underwent rRNA deleting through RNAcentral to get effective reads. Genomic alignment (version from UCSC genome browser) was using Tophat(version:2.0.13) to get uniquely mapping reads.
Differentially expressed genes
RNAs differential expression analysis was performed by DESeq2 software between two different groups (and by edgeR between two samples). The genes/transcripts with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change≥2 were considered differentially expressed genes/transcripts.
Pathway enrichment analysis
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using KOBAS3.0. The results from the enrichment analysis were restricted to KEGG pathway terms, with adjusted P-value < 0.05 considered to be significant. Pathway significance enrichment analysis using KEGG Pathway as unit, hypergeometric test was applied to find out that compared with background genes, Pathway of significant enrichment in differentially expressed genes. After multiple test correction, Pathway with Qvalue ≤ 0.05 was defined as significantly enriched in differentially expressed genes. The Pathway. Q-value here is the P-value after FDR correction. The involvement of differentially expressed genes could be identified through significant enrichment of Pathway. The most important biochemical metabolic pathway and signal transduction pathway.
Western blot analysis
Cultured cells and mouse brain tissues were homogenized in lysis buffer (RIPA) in the presence of protease inhibitor (PMSF) for 30 min on ice and centrifuged at 12000 rpm for 30 min at 4 °C. Protein samples (30–60 μg) were separated on 10% SDS-PAGE gels and then immunoblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore) in ice-cold buffer (25 mM Tris-HCl, 192 mM glycine and 20% methanol) by electrotransfer for 2 h. The membranes were blocked with 5% non-fat milk powder in TBST and then incubated with the indicated antibodies at 4 °C overnight (GRIN2B, Immunoway: YT3152, 1:1000; GRIA1, Immunoway: YM8615, 1:1000; PKC, Immunoway: YM4217, 1:1000; NEDD4L, Immunoway: YN3050, 1:1000; PSD95, Immunoway: YM8292, 1:1000; GAPDH, Proteintech: 60004, 1:5000). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Abclonal: AS014, 1:5000) and goat anti-mouse (Abclonal: AS003, 1:5000) secondary antibodies were incubated at room temperature for 1 h. Blots were detected by enhanced chemiluminescence (Pierce). Protein abundance was quantified by analyzing Western blot bands using Image Lab software (Bio-Rad). Quantified band intensities were normalized to GAPDH levels and averaged from at least three independent experiments.
Real-time quantitative PCR
The total RNA from the whole blood and brain tissues was extracted using TRIzol (Thermo Fisher Scientific) in line with the manufacturer’s instructions, and the RNA samples were stored at −80 °C. Quantitative PCR was performed by using the Transcriptor First Strand cDNA Synthesis kit (TAKARA) and TB Green® Premix Ex Taq™ II (TAKARA) on a LightCycler 96 Real-Time System (Roche). Differences in gene expression were calculated by the 2-ΔΔCT method and were presented as the fold change. The relevant primers were provided in the Supplementary Table 1 in Supplementary Material.
Immunofluorescence staining
Animals were anesthetized and perfused with saline followed by 4% PFA in 0.1 M PBS, pH 7.4. The brains of the animals were removed, post-fixed overnight in 4% PFA at 4 °C and transferred to 30% sucrose in 0.1 M PBS, pH 7.4, followed by cutting the coronal sections (40 μm) on a freezing microtome (Leica CM3050 S). And permeabilized in 0.5% Triton X-100 solution for 5 min at room temperature. Then, the cells and sections were washed with PBS for 3 times and incubated first in blocking buffer containing 3% bovine serum albumin in 0.2% Triton X-100/PBS for 2 h at room temperature and then with primary antibodies against GRIA1 (1:500; Immunoway: YM8615), PSD95 (1:500; Immunoway: YM8292) in blocking buffer overnight at 4 °C. After washing the treated sections with PBS again for three times, they were further incubated with Alexa Fluor 647- or Alexa Fluor 568-conjugated secondary antibodies at room temperature for 2 h. Alexa fluor 647-anti-Rabbit secondary antibody (Immunoway, RS3811, 1:500), Alexa fluor 568-anti-Rabbit secondary antibody (Immunoway, RS3511, 1:500). The nuclei were counterstained with DAPI and the coverslips were mounted onto glass slides with anti-fade solution and visualized using a Nikon fluorescence microscope (Nikon Instruments Inc.).
Electrophysiological recordings
Mice were anaesthetized with pentobarbital sodium. Their brains were rapidly removed and sliced into 300 μm horizontal slices containing mPFC using a Leica ls1200 vibratory slicer. They were prepared in cryogenically sliced artificial cerebrospinal fluid (aCSF) containing (mM) 220 sucrose, 2.5 KCl, 1.3 CaCl2, 2.5 MgSO4, 1 NaH2PO4, 26 NaHCO3 and 10 glucose. The sections were then transferred to a chamber containing oxygenated aCSF (in millimetres) of 126 NaCl, 26 NaHCO3, 3.0 KCl, 1.2 NaH2PO4, 2.0 CaCl2, 1.0 MgSO4 and 10 glucose and stored at 32 °C for 30 min followed by 1 h at room temperature. The mPFC pyramidal neurons were observed using a 40x water immersion objective (Nikon). Recording electrodes with a resistance of −5 MΩ were removed from borosilicate glass capillaries. To record ministure excitatory postsynaptic currents (mEPSCs), the internal pipette solution for the recording electrodes contained (in mM) 125 cesium methanesulfonate, 5 CsCl, 10 HEPES, 0.2 EGTA, 1 MgCl2, 4 Mg-ATP, 0.3 Na-GTP, 10 phosphocreatine and 5 QX314 (pH 7.40, 290 mOsm). Recordings were made with a HEKA EPC10 amplifier with the signals filtered at 5 kHz and digitized at 10 kHz. The mEPSCs with 20 μM bicuculine (Sigma) and 1 μM tetrodotoxin (Aladdin) added to the artificial cerebrospinal fluid in the voltage clamp (Vclamp = −70 mV).
Virus injection
Eight-week-old mice were anaesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal injection) and fixed in a stereotaxic frame (RWD) prior to surgery. They were then treated with a bilateral stereotaxic injection of the virus into the mPFC (AP: +1.75; ML: ±0.3; DV: −2.7 mm relative to the bregma. AP, ML and DV denote anteroposterior, mediolateral and dorsoventral distances from the bregma, respectively. These coordinates were measured from the bregma according to the mouse atlas. The virus (300 nL) was injected into each location at a rate of 100 nL per min. After each injection, the needle was left in place for six minutes and then slowly withdrawn.
The viruses rAAV2/9-hSyn-EGFP-5’miR-30a-shRNA(Scramble)- 3’miR30a WPREs and rAAV2/9- hSyn-EGFP-5’miR-30a-shRNA(Grin2b)- 3’miR30a WPREs were purchased from BrainVTA (Wuhan, China). The shRNA sequences targeting Grin2b were GCAGCAATATAAGGACAGTCTA.
Mouse brain slice preparation
Mice were anesthetized and perfused with saline followed by 4% PFA in 0.1 M PBS, pH 7.4. The brains of the animals were removed, post-fixed overnight in 4% PFA at 4 °C and transferred to 30% sucrose in 0.1 M PBS, pH 7.4, followed by cutting the coronal sections (40 μm) on a freezing microtome (Leica CM3050 S). The nuclei were counterstained with DAPI and the coverslips were mounted onto glass slides with anti-fade solution and visualized using a Nikon fluorescence microscope (Nikon Instruments Inc.).
Quantification and statistical analysis
All experiments and data analyses were performed in a blinded fashion. Statistical comparisons were performed using SPSS 20.0 software with appropriate inferential methods, with t-data presented as mean ± s.e.m. Normally distributed data were tested by two-tailed unpaired t-test for two-group comparisons and by one- and two-way analysis of variance (ANOVA) followed by Bonferroni’s test for multiple comparisons. Non-normally distributed data were analyzed using Mann-Whitney U tests for two group comparisons and Kruskal-Wallis test and Dunn’s multiple comparison test for more than two groups. Statistical significance was set at *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. ns, no significance.
Results
Perampanel treatment rapidly alleviates depression-like behavior in CSDS-susceptible mice
Previous studies have demonstrated that a single dose of 0.5 mg/kg ketamine upregulates the expression of the Gria1 subunit of AMPAR in the mPFC [26, 29]. The elevated Gria1 expression is considered a proposed mechanism underlying the antidepressant effects of ketamine [18, 19]. To investigate whether Gria1 expression levels are altered in the mPFC of depression model mice, we produced CSDS depression susceptible mice (Sus, SI ratio<1) (Fig. 1A-B, Figure S1A). Then, we administered a single injection of 0.5 mg/kg of perampanel-treated Sus (SP0.5) after 1 h (Fig. 1C). The western blotting analysis showed that the expression level of total Gria1 was no difference in the Sus group mice compared to Naïve group mice in the mPFC (Fig. 1D). This result is consistent with previous studies [30]. However, the western blotting analysis showed that the expression level of Gria1 was increased in the SP0.5 group mice in the mPFC (Fig. 1D). In the social interaction test (SI), Sus group mice showed significantly decrease of SI ratio compared to Naïve group mice. SP0.5 group mice showed significantly increase of the SI ratio compared to Sus group mice, and no difference with Naïve group (Fig. 2E, Figure S1C). In the FST and the TST, Sus group mice showed significantly increase of the immobility time compared to Naïve group mice. Mice in the SP0.5 group exhibited a significant decrease in immobility time compared to those in the Sus group, while no significant difference was observed compared to the Naïve group (Fig. 1F-G). These results indicate that a single 0.5 mg/kg injection of perampanel elicits rapid antidepressant effects in stress-susceptible mice.
A Schematic of the CSDS paradigm. B Social interaction ratio in social interaction test of Naïve, Res and Sus group mice. (Naïve: n = 6; Res: n = 15; Sus: n = 15, Brown-Forsythe and Welch ANOVA tests: Naïve vs. Res p = 0.9991, Naïve vs. Sus ****p < 0.0001, Res vs. Sus ****p < 0.0001). C Schematic of perampanel injection after 1 h and behavioral testing. D Western blotting analysis of the Gria1 in mPFC of Naïve, Sus and SP0.5 group. (Naïve: n = 6; Sus: n = 6; SP0.5: n = 5, one-way ANOVA: Naïve vs. Sus, p > 0.9999, Naïve vs. SP0.5, ***p = 0.0004, Sus vs. SP0.5, **p = 0.0014; F (2, 14) = 15.75). E Social interaction ratio in social interaction test of Naïve, Sus and SP0.5 group mice. (Naïve: n = 6; Sus: n = 8; SP0.5: n = 7, one-way ANOVA: Naïve vs. Sus, ***p = 0.0002, Naïve vs. SP0.5, p = 0.2449, Sus vs. SP0.5, **p = 0.0086; F (2, 18) = 14.32). F Immobility time in force swimming test of Naïve, Sus and SP0.5 group mice. (Naïve: n = 6; Sus: n = 8; SP0.5: n = 7, one-way ANOVA: Naïve vs. Sus, *p=0.0140, Naïve vs. SP0.5, p > 0.9999, Sus vs. SP0.5, **p = 0.0062; F (2, 18) = 8.108). G Immobility time in tail suspension test of Naïve, Sus and SP0.5 group mice. (Naïve: n = 6; Sus: n = 8; SP0.5: n = 7, one-way ANOVA: Naïve vs. Sus, *p = 0.0275, Naïve vs. SP0.5, p > 0.9999, Sus vs. SP0.5, *p = 0.0172; F (2, 18) = 6.353). H Total distances in open field test of Naïve, Sus and SP0.5 group mice. (Naïve: n = 6; Sus: n = 8; SP0.5: n = 7, one-way ANOVA: Naïve vs. Sus, p > 0.9999, Naïve vs. SP0.5, p > 0.9999, Sus vs. SP0.5, p = 0.8417; F (2, 18) = 0.6376).
A RNA sequencing schematic. B Percentage of differential genes that are up and down in Naïve vs. Sus, Sus vs. SP0.5, Naïve vs. SP0.5. C Wayne diagrams show DiffExp genes intersection of Naïve vs. Sus, Sus vs. SP0.5, Naïve vs. SP0.5. D Volcano plot showing the number of differential genes that up and down in Naïve vs. Sus, Sus vs. SP0.5, Naïve vs. SP0.5. E KEGG enrichment of nervous system-associated annotations in terms of organismal system analysis. F Wayne diagrams show KEGG enrichment of nervous system-associated annotations intersection of Naïve vs. Sus, Sus vs. SP0.5, Naïve vs. SP0.5. G Wayne diagrams show intersection of DiffExp genes and KEGG. H Heat map of 14 differential gene expression.
Meanwhile, no difference in locomotor performance in the open field test (OFT) of Naïve, Sus and SP0.5 groups, the Sus group mice showed significantly decrease of the center time compared to Naïve group mice. SP0.5 group mice showed significantly increase of the center time compared to Sus group mice, and no difference with Naïve group (Fig. 1H, Figure S1B). After mice were exposed to anxiety-related behavioral tests, SP0.5 group mice spent less time in the closed arms and more time in the open arms of elevated plus maze (EPM) compared to Sus group mice, and no difference with Naïve group (Figure S1D). Together, these data suggest that a single 0.5 mg/kg injection of perampanel in CSDS-susceptible mice produces rapid antidepressant and anxiolytic effects. Changes in Gria1 expression may be the main reason for the rapid antidepressant effect, as previously reported [18, 19].
Perampanel reduces Grin2b mRNA abnormal expression in stress-susceptible mice
Perampanel is a non-competitive AMPAR antagonist previously reported to downregulate Gria1 expression in brain tissue of epilepsy models [24]. Here, however, we observed a contrasting effect in Sus mice, where perampanel administration significantly increased Gria1 levels specifically in the mPFC and concurrently produced antidepressant effects. To resolve this paradox and identify the transcriptional basis for its therapeutic action, we conducted mRNA sequencing on mPFC samples from Naïve, Sus, and SP0.5 mice, followed by differential gene expression analysis (Fig. 2A-B, Figure S2A). We screened 14 genes that were intersected by genes significantly differentially expressed in Naïve versus Sus and genes significantly differentially tabulated in Sus versus SP0.5 and were not within the differential gene range of Naïve versus SP0.5 (Fig. 2C). Then, we clarified the elevated or reduced expression of these 14 genes in the differential gene volcano plot and found that among these 14 genes Grin2b expression was most significantly elevated in the mPFC of Sus mice compared to the Naïve group. Administration of perampanel (0.5 mg/kg) not only significantly reduced Grin2b expression in the SP0.5 group compared to the Sus group, but, importantly, brought it to a level that was not statistically different from the Ctrl group (Fig. 2D).
Next, we enriched to differential gene expression in the nervous system using KEGG annotation (Fig. 2E) and further screened the genes that were significantly differentially expressed in Naïve versus Sus and intersected with the genes that were significantly differentially tabulated in Sus versus SP0.5 and were not within the range of genes that were differentially expressed in Naïve versus SP0.5 for 1 gene (Fig. 2F). The Grin2b gene, which is present in both, was identified by combining differential genes with KEGG enrichment (Fig. 2G). Interestingly, the mRNA expression of Grin2b—a subunit of the NMDAR—was significantly elevated in the Sus group compared to the Naïve group. This elevated expression was concurrently reversed by a 0.5 mg/kg dose of perampanel, as evidenced by the significant reduction in the SP0.5 group compared to the Sus group (Fig. 2H). Meanwhile, we performed real-time quantitative PCR (qRT-PCR) to analyze the mRNA expression of Gria1 and Grin2b. The mRNA expression of Gria1 showed no significant difference in the Sus group mice compared to the Naïve group mice in the mPFC. However, it was significantly increased in the SP0.5 group mice injected with 0.5 mg/kg perampanel, which is consistent with the previously observed changes in Gria1 protein expression. In contrast, the mRNA expression of Grin2b was significantly elevated in the mPFC of Sus group mice compared to the Naïve group. Nevertheless, administration of 0.5 mg/kg perampanel in the SP0.5 group significantly reduced Grin2b mRNA expression relative to the Sus group (Figure S2B-C). We therefore conclude that perampanel normalizes CSDS-induced Grin2b upregulation. These results show that reduced expression of Grin2b may be the main reason for the rapid antidepressant effect of perampanel in Sus mice.
Perampanel has different effects in stress-susceptible and Naïve mice
To validate the mRNA sequencing results of Grin2b, we analyzed the western blot results are show that the Grin2b expression was significantly elevated in the mPFC of Sus mice compared to the Naïve group. Meanwhile, after administration of perampanel at 0.5 mg/kg the Grin2b expression of SP0.5 group was significantly reduced compared to the Sus group (Fig. 3A). This result is consistent with mRNA sequencing and qRT-PCR results of Grin2b. We then compared the basal Grin2b/GluA1 ratio in the mPFC between Naïve and Sus mice prior to treatment. A comparison of the basal Grin2b/GluA1 ratio in the mPFC revealed a preexisting difference between Naïve and Sus mice (Fig. 3B), and this identified baseline discrepancy may be critical to perampanel’s efficacy in reversing depression-like behaviors in the Sus group.
A Western blotting analysis of the Gri2B in mPFC of Naïve, Sus and SP0.5 group. (Naïve: n = 6; Sus: n = 5; SP0.5: n = 5, one-way ANOVA: Naïve vs. Sus, **p = 0.0055, Naïve vs. SP0.5, p > 0.9999, Sus vs. SP0.5, **p = 0.0031; F (2, 13) = 10.84). B Basal mPFC Grin2b/GluA1 expression in naïve vs. Sus mice pre-treatment. C Schematic of perampanel injection after 1 h and behavioral testing. D Western blotting analysis of the Gria1 in mPFC of Naïve and NP0.5 group. (Naïve: n = 4; NP0.5: n = 4, unpaired t test: *p = 0.0325, t = 2.769). E Western blotting analysis of the Grin2b in mPFC of Naïve and NP0.5 group. (Naïve: n = 4; NP0.5: n = 4, unpaired t test: **p = 0.0042, t = 4.481). F Social interaction ratio in social interaction test of Naïve and NP0.5 group mice. (Naïve: n = 6; NP0.5: n = 6, welch’s t test: *p = 0.0105, t = 3.796). G Immobility time in force swimming test of Naïve and NP0.5 mice. (Naïve: n = 6; NP0.5: n = 6, unpaired t test: **p = 0.0074, t = 3.345). H Immobility time in tail suspension test of Naïve and NP0.5 mice. (Naïve: n = 6; NP0.5: n = 6, unpaired t test: **p = 0.0068, t = 3.397). I Total distances in open field test of Naïve and NP0.5 group mice. (Naïve: n = 6; NP0.5: n = 6, unpaired t test: **p = 0.0068, t = 3.397). J Schematic diagram of conditioned place preference; Preference ratio in conditioned place preference of Naïve and NP0.5 group mice. (Naïve: n = 9; NP0.5: n = 9, paired t test, p = 0.3867, t = 0.9480). K Percentage of prepulse inhibition of the auditory startle reflex across different prepulse intensities. (Naïve: n = 6; NP0.5: n = 6, unpaired t test: The auditory startle reflex across different prepulse intensities, unpaired t test: 74 dB, p = 0.8901, t = 0.1418; 78 dB, p = 0.4322, t = 0.8183; 86 dB, p = 0.4824, t = 0.7295; 82 dB, p = 0.5523, t = 0.6144; 90 dB, p = 0.9163, t = 0.1078).
To further comprehensively evaluate the effects of perampanel on depression, we administered a single injection of the same dose of perampanel into Naïve mice (NP0.5) after 1 h, (Fig. 3C). The western blotting analysis showed that the expression level of Gria1 was decrease in the NP0.5 group mice in the mPFC (Fig. 3D), as previously reported. We also found reduced Grin2b expression levels in Naïve mice injected with perampanel in the NP0.5 group compared to the Naïve group (Fig. 3E). These results indicate that Grin2b expression decreased in the NP0.5 mice, while total Gria1 expression levels remained decrease compared to Ctrl mice, as previously reported. For behavioral studies, we first investigated the behavioral performance of NP0.5 mice and littermate controls. In the SI, the social interaction ratio was decreased in NP0.5 mice compared with Ctrl mice (Fig. 3F). In the FST, the duration of immobility was increased in NP0.5 mice (Fig. 3G). In the TST, the duration of immobility was increased in NP0.5 mice (Fig. 3H). These results show that a single 0.5 mg/kg injection of perampanel rapidly induces depressive-like behavior in mice.
Meanwhile, NP0.5 group mice showed no difference in locomotor performance in the OFT, the center time was decreased in NP0.5 mice compared with Ctrl mice (Fig. 3K, Figure S3A). After mice were exposed to anxiety-related behavioral tests, NP0.5 group mice spent more time in the closed arms of EPM (Figure S3B). To further investigate whether a single injection of 0.5 mg/kg perampanel causes addiction and hallucinations, we performed CPP and PPI behavioral tests. CPP results showed no difference in preference ratios between the NP0.5 and Naïve groups (Fig. 3I). In PPI test, no difference in prepulse inhibition percentage between the NP0.5 and Naïve groups (Fig. 3J). Together, these data suggest that a single 0.5 mg/kg injection of perampanel in mice produces rapid depressive and anxious behavior without leading to locomotor abnormalities, addiction or hallucinations.
Perampanel exerts rapid antidepressant effects via reducing Grin2b expression
Sus mice exhibited increased Grin2b expression levels while no difference of total Gria1 expression levels compared to Naïve group. Whereas Grin2b and total Gria1 expression decreased in the NP0.5 mice. Both Sus mice and NP0.5 mice exhibited depression-like behavior. To investigate whether changes in Grin2b expression contribute to the antidepressant effects of perampanel, we first established a cohort of stress-susceptible mice, defined by a SI ratio of less than 1 (Figure S4A). Then, we specifically knockdown Grin2b in the mPFC of Sus group mice by using bilateral injection of rAAV2/9-hSyn-EGFP-shRNA(Grin2b) (shRNA virus) or rAAV2/9- hSyn-EGFP (Ctrl virus) (Fig. 4A). Confocal imaging and Western blotting analysis showed that viral transfection resulted in reduced Grin2b expression in the mPFC neurons (Fig. 4B-C). For behavioral studies, In the SI, Sus + Ctrl virus + saline group mice (SCS) showed significantly decrease of SI ratio compared to Naïve + Ctrl virus + saline group mice (NCS). Sus + shRNA virus + saline group mice (SshS) showed significantly increase of the SI ratio compared to SCS group mice, and no difference with NCS group (Fig. 4D-F). In the FST and in the TST, SCS group mice showed significantly increase of the immobility time compared to NCS group mice. SshS group mice showed significantly decrease of the immobility time compared to SCS group mice (Fig. 4D-F). At this time, the Western blotting analysis showed that Gria1 expression increased in the SshS group compared to the SCS group (Fig. 4C). However, there was no significant difference in Gria1 expression between the SCS and NCS groups (Fig. 4C). These results suggest that reduced expression of Grin2b can directly alleviate depressive-like behavior in CSDS-susceptible mice, it also increases the expression level of Gria1.
A Schematic of perampanel injection after 1 h and behavioral testing. B Representative diagram of adenovirus injection. C Western blotting analysis of the Gri2B and Gria1 in mPFC of NCS, SCS, SshS, NshS and NshP group. (Grin2b: NCS: n = 3; SCS: n = 3; SshS: n = 3; NshS: n = 3; NshP: n = 3, NCS vs. SCS, welch’s t test, *p = 0.0189, t = 7.171; SCS vs. SshS, unpaired t test, *p = 0.0157, t = 4.030; NCS vs. NshS, welch’s t test, *p = 0.0176, t = 7.448; NshS vs. NshP, unpaired t test, *p = 0.0126, t = 4.305. Gria1: NCS: n = 3; SCS: n = 3; SshS: n = 3; NshS: n = 3; NshP: n = 3, NCS vs. SCS, welch’s t test, p = 0.6985, t = 0.4472; SCS vs. SshS, unpaired t test, *p = 0.0445, t = 2.891; NCS vs. NshS, welch’s t test, *p = 0.0205, t = 6.880; NshS vs. NshP, unpaired t test, *p = 0.0347, t = 3.146). D Social interaction ratio in social interaction test of NCS, SCS, SshS, NshS and NshP group. (NSC: n = 8; SCS: n = 6; SshS: n = 6; NshS: n = 6; NshP: n = 6, NCS vs. SCS, welch’s t test, *p = 0.0415, t = 2.395; SCS vs. SshS, unpaired t test, **p = 0.0097, t = 3.189; NCS vs. NshS, unpaired t test, p = 0.5944, t = 0.5469; NshS vs. NshP, unpaired t test, p = 0.5236, t = 0.6610). E Immobility time in force swimming test of NCS, SCS, SshS, NshS and NshP group. (NCS: n = 8; SCS: n = 6; SshS: n = 6; NshS: n = 6; NshP: n = 6, NCS vs. SCS, unpaired t test, *p = 0.0106, t = 3.023; SCS vs. SshS, unpaired t test, **p = 0.0047, t = 3.619; NCS vs. NshS, unpaired t test, p = 0.8263, t = 0.2243; NshS vs. NshP, unpaired t test, p = 0.8943, t = 0.1363). F Immobility time in tail suspension test of NCS, SCS, SshS, NshS and NshP group. (NCS: n = 8; SCS: n = 6; SshS: n = 6; NshS: n = 6; NshP: n = 6, NCS vs. SCS, unpaired t test, **p = 0.0086, t = 3.137; SCS vs. SshS, unpaired t test, *p = 0.0263, t = 2.605; NCS vs. NshS, unpaired t test, p = 0.6434, t = 0.4749; NshS vs. NshP, unpaired t test, p = 0.7086, t = 0.3845). G Total distances in open field test of NCS, SCS, SshS, NshS and NshP group. (NCS: n = 8; SCS: n = 6; SshS: n = 6; NshS: n = 6; NshP: n = 6, NCS vs. SCS, unpaired t test, p = 0.7699, t = 0.2993; SCS vs. SshS, unpaired t test, p = 0.5729, t = 0.5829; NCS vs. NshS, unpaired t test, p = 0.5214, t = 0.6606; NshS vs. NshP, unpaired t test, p = 0.5559, t = 0.6093).
Meanwhile, no difference in locomotor performance in the OFT of NCS, SCS and SshS groups, the SCS group mice showed significantly decrease of the center time compared to NCS group mice. SshS group mice showed no difference of the center time compared to SCS group mice (Fig. 4G, Figure S4B). SshS group mice no difference with SCS group of spent time in the closed arms and open arms of EPM (Figure S4C). Together, these data suggest that reducing Grin2b expression in the mPFC of CSDS-susceptible mice improves depressive-like behavior, but not anxiety-like behavior. A single injection of 0.5 mg/kg of perampanel produces a rapid anxiolytic effect that does not appear to be mediated by reduced Grin2b expression in the mPFC.
Secondly, to further investigate whether the reduced expression of Grin2b in the mPFC of NP0.5 mice is the cause of depressive-like behavior. We specifically knockdown Grin2b in the mPFC of Naïve group mice by using bilateral injection of rAAV2/9-hSyn-EGFP-shRNA(Grin2b) (shRNA virus) or rAAV2/9- hSyn-EGFP (Ctrl virus) (Fig. 4A). Confocal imaging and Western blotting analysis showed that viral transfection resulted in reduced Grin2b expression in the mPFC neurons (Fig. 4B-C). For behavioral studies, In the SI, Naïve + shRNA virus + saline group mice (NshS) showed no difference of SI ratio compared to Naïve + Ctrl virus + saline group mice (NCS). In the FST and the TST, NshS group mice showed no difference of the immobility time compared to NCS group mice. (Fig. 4D-F). These results suggest that reduced expression of Grin2b alone cannot induce depressive-like behavior in Naïve mice.
At this time, the western blotting analysis showed that Gria1 expression increase in the NshS group compared to the NCS group (Fig. 4C). Meanwhile, there was decrease in Gria1 expression in the NshP group compared to the NshS group (Fig. 4C). The knockdown of Grin2b results in elevated expression of Gria1 in the mPFC of Naïve mice. NshP mice have been observed to exhibit a further reduction in levels of Grin2b expression; however, this Grin2b reduction caused by perampanel does not result in further increases in Gria1. In the context of Naïve mice, Gria1 expression in the mPFC of NshP mice exhibits a reduction that is analogous to that observed in Naïve mice, in comparison to NshS mice. This finding aligns with the observations presented in Fig. 3C-D. These results suggest that perampanel exerts divergent effects on the regulation of Gria1 expression in Sus and Naïve mice. Next, in the social interaction test (SI), Naïve + shRNA virus + perampanel group mice (NshP) showed no difference of SI ratio compared to Naïve + shRNA virus + saline group mice (NshS). In the FST and the TST, NshP group mice showed no difference of the immobility time compared to NshS group mice. (Fig. 4D-F). These results indicate that the simultaneous reduction of Grin2b and total Gria1 induced by perampanel is critical for depressive-like behavior in Naïve mice.
Meanwhile, no difference in motor performance was observed between the NCS, NshS and NshP groups in the OFT. However, mice in the NshP group exhibited significantly reduced center time compared to those in the NshS group. There was no difference in center time between the NshS and NCS groups (Fig. 4G, Figure S4B). In the EPM, mice in the NshP group spent significantly more time in the closed arms and less time in the open arms in contrast to the NCS group, whereas the NshS group showed no significant difference from the NCS controls (Figure S4C). These results demonstrate that changes in Grin2b expression in the mPFC are not associated with the perampanel-affected anxiety-like behaviors. Taken together, these data indicate that reducing Grin2b expression in the mPFC of Naïve mice is not the underlying cause of depressive-like behavior in NP0.5 mice, and that further administration of 0.5 mg/kg perampanel does not induce depressive-like behavior, but still induces anxiety-like behavior.
Grin2b knockdown in susceptible mice increases Gria1 by regulating relevant enzymes
Numerous classical enzymatic pathways, involving phosphorylation and ubiquitination, have been reported to regulate the expression and membrane trafficking of AMPA receptor subunits [31,32,33,34,35]. To elucidate the mechanism by which perampanel reduces Grin2b expression and subsequently upregulates Gria1 in Sus mice, we first specifically knocked down Grin2b in the mPFC of Sus group mice via bilateral injection of rAAV2/9-hSyn-EGFP-shRNA(Grin2b) (designated as the Sus-shRNA(Grin2b) group) or a control virus, rAAV2/9-hSyn-EGFP (Sus group). We then compared the gene expression of enzymes potentially influencing Gria1 expression between these groups.
Our results revealed that the phosphorylation-related gene Prkca (encoding PKCα, a kinase known to regulate AMPA receptor function and synaptic plasticity) [36,37,38] was significantly increased upon Grin2b knockdown. Conversely, the ubiquitination-related gene Nedd4l (encoding the NEDD4-2 E3 ubiquitin ligase, which mediates the ubiquitination and degradation of AMPA receptors) [35, 39,40,41] was significantly decreased (Fig. 5A). Western blot analysis confirmed that, compared to the Sus group, the Sus-shRNA(Grin2b) group exhibited reduced Grin2b and Nedd4l protein levels, alongside elevated Gria1 and Prkca protein expression. Furthermore, we observed a significant increase in the expression of PSD-95, providing a structural basis for enhanced AMPA receptor synaptic localization [42,43,44] (Fig. 5B-G).
A mRNA expression of enzyme-related genes (Sus vs. Sus-shRNA(Grin2b), n = 3, unpaired t test, Prkca: *p = 0.0381, t = 3.048; welch’s t test, Nedd4l: *p = 0.0403, t = 4.773). B Representative Western blot images of Sus and Sus-shRNA(Grin2b) group (Grin2b, Gria1, Prkca, Nedd4l, PSD-95, GAPDH). C Western blot analysis of Grin2b in Sus and Sus-shRNA(Grin2b) group (Sus vs. Sus-shRNA(Grin2b), n = 6, unpaired t test, *p = 0.0140, t = 3.253). D Western blot analysis of Gria1 in Sus and Sus-shRNA(Grin2b) group (Sus vs. Sus-shRNA(Grin2b), n = 6, unpaired t test, *p = 0.0386, t = 2.381). E Western blot analysis of Prkca in Sus and Sus-shRNA(Grin2b) group (Sus vs. Sus-shRNA(Grin2b), n = 6, unpaired t test, *p = 0.0280, t = 2.569). F Western blot analysis of Nedd4l in Sus and Sus-shRNA(Grin2b) group (Sus vs. Sus-shRNA(Grin2b), n = 6, unpaired t test, *p = 0.0447, t = 2.294). G Western blot analysis of PSD-95 in Sus and Sus-shRNA(Grin2b) group (Sus vs. Sus-shRNA(Grin2b), n = 6, unpaired t test, *p = 0.0220, t = 2.708). H Representative immunofluorescence images of Sus and Sus-shRNA(Grin2b) group, Scale bar, 100 µm. I Immunofluorescence Counting of PSD-95 (Sus vs. Sus-shRNA(Grin2b), n = 5, unpaired t test, **p = 0.0018, t = 4.593). J Immunofluorescence Counting of Gria1 (Sus vs. Sus-shRNA(Grin2b), n = 5, unpaired t test, *p = 0.0273, t = 2.694). K Immunofluorescence Counting of merge of PSD-95 and Gria1 (Sus vs. Sus-shRNA(Grin2b), n = 5, unpaired t test, **p = 0.0050, t = 3.830).
To further determine the subcellular distribution of the increased Gria1, we performed immunofluorescence co-staining for PSD-95 and Gria1. We found that not only were the individual expression levels of PSD-95 and Gria1 increased in the Sus-shRNA(Grin2b) group, but their co-localization was also significantly enhanced (Fig. 5H-K). Collectively, these findings indicate that knocking down Grin2b expression in the mPFC of Sus mice leads to a significant increase in postsynaptic Gria1 expression, mechanistically driven by the upregulation of Prkca and downregulation of Nedd4l. This augmentation of Gria1 at the postsynaptic membrane is likely a key factor underlying the subsequent alterations in excitatory synaptic transmission.
Perampanel regulates neuronal excitatory synaptic transmission
The function of neuronal excitatory synaptic transmission is affected by changes in the expression levels of glutamate receptor subunits. It is well-established that an increase in postsynaptic AMPA receptors can feedback to enhance presynaptic glutamate release [45,46,47,48,49]. To investigate whether the observed increase in synaptic Gria1 expression, resulting from reduced Grin2b in the mPFC of Sus mice, alters presynaptic glutamate release and consequently affects neuronal excitatory synaptic function, we recorded miniature excitatory postsynaptic currents (mEPSC) in mPFC pyramidal neurons from mice in the Naïve, Sus, SP0.5 and NP0.5 groups, respectively. The mEPSC results show that the mEPSC frequency of mPFC pyramidal neurons in the Sus group was significantly decreased, but the mEPSC amplitude was not significantly altered compared to the Naïve group (Fig. 6A-C). Meanwhile, the mEPSC frequency of mPFC pyramidal neurons in the SP0.5 group injected with perampanel had recovered, with no significant change in mEPSC amplitude compared to the Sus group (Fig. 6A-C). These results suggest that the increased postsynaptic Gria1 expression, caused by reduced Grin2b, enhances excitatory neurotransmitter release from the presynaptic membrane in the mPFC of Sus mice, thereby rescuing the deficit in excitatory synaptic transmission.
A Schematic diagram and representative diagram for mEPSC recording of Naïve, Sus, SP0.5 and NP0.5 group. B Frequency distribution and statistical analysis of mEPSC in Naïve, Sus, SP0.5 and NP0.5 group. (Naïve: n = 6; Sus: n = 6; SP0.5: n = 11; NP0.5: n = 14, one-way ANOVA: Naïve vs. Sus, *p = 0.0111, Naïve vs. SP0.5, p > 0.9999, Sus vs. SP0.5, **p = 0.0036, Naïve vs. NP0.5, ***p = 0.0003; F (3, 33) = 14.61). C Amplitude distribution and statistical analysis of mEPSC in Ctrl, Sus and PER0.5 group. (Naïve: n = 6; Sus: n = 6; SP0.5: n = 11; NP0.5: n = 14, one-way ANOVA: Naïve vs. Sus, p > 0.9999, Naïve vs. SP0.5, p > 0.9999, Sus vs. SP0.5, p = 0.3043, Naïve vs. NP0.5, ****p < 0.0001; F (3, 33) = 61.21).
However, the mEPSC results show that the mEPSC frequency and amplitude of mPFC pyramidal neurons in the NP0.5 group was significantly decreased compared to the Naïve group (Fig. 6A-C). These results suggest that perampanel reduces excitatory synaptic transmission in neurons in Naïve mice. Meanwhile, the inhibitory effect of perampanel on mEPSC amplitude accounts for the absence of an increase in mEPSC amplitude in the SP0.5 group, despite the elevated postsynaptic Gria1 expression in the mPFC. Together, these data show that perampanel rescued the function of neuronal excitatory synaptic transmission by indirectly decreasing Grin2b expression in Sus mice.
Perampanel’s antidepressant efficacy lasts for 12 h in mice
In clinical practice, it has been shown that perampanel has a long half-life [50, 51]. Thus, we evaluated the antidepressant effects of a single 0.5 mg/kg dose of perampanel in CSDS depression susceptible mice to 12 h after injection (Fig. 7A-C, Figure S5A). After 12 h, in the SI, Sus group mice showed significantly decrease of SI ratio compared to Naïve group mice. SP0.5 group mice showed no difference of the SI ratio compared to Sus group mice, and decrease compared to Naïve group (Fig. 7D). However, at this point, a minority of the SP0.5 group of mice still had an SI ratio >1 (Fig. 7E). Therefore, we compared the SI ratios of the mice in the SP0.5 group by pairs, pre and post 12 h of perampanel injection. SI results showed that the SI ratio of the mice in the SP-post group was significantly higher than that of the mice in the SP-pre group (Fig. 7F-G). No difference in motor performance was observed between the Naïve, Sus, SP0.5 groups and SP-pre, SP-post groups in the OFT (Figure S5B-C). Collectively, these data suggest that the prolonged half-life of perampanel contributes to the maintenance of its antidepressant effects.
A Schematic of the CSDS paradigm. B Social interaction ratio in social interaction test of Naïve, Res and Sus group mice. (Naïve: n = 8; Res: n = 9; Sus: n = 18, one-way ANOVA: Naïve vs. Res p = 0.7954, Naïve vs. Sus ****p < 0.0001, Res vs. Sus ****p < 0.0001; F (2, 32) = 37.82). C Schematic of perampanel injection after 12 h and SI behavioral testing. D Social interaction ratio in social interaction test of Naïve, Sus and SP0.5 group mice. (Naïve: n = 8; Sus: n = 9; SP0.5: n = 9, one-way ANOVA: Naïve vs. Sus, ***p = 0.0003, Naïve vs. SP0.5, **p = 0.0044, Sus vs. SP0.5, p = 0.7448; F (2, 23) = 12.16). E Pie chart of SI ratio. F Schematic of perampanel injection after 12 h and SI behavioral testing of SP-pre and SP-post. G Social interaction ratio in social interaction test of SP-pre and SP-post group mice. (SP-pre: n = 9; SP-post: n = 9, paired t test, **p = 0.0063, t = 3.666).
Discussion
Our study reveals another potential antidepressant drug that targets the glutamate system. It produces rapid, long-lasting antidepressant effects without inducing addiction or hallucinations. Perampanel is an FDA-approved medication for the treatment of epilepsy. The new use of this medication has the potential to benefit patients with depression and patients with epilepsy and depression. However, it should be used with caution in patients with simple epilepsy. This opens up new possibilities and provides biological guidance for the translational application of clinical neurological medications.
Major depressive disorder is a serious mental illness affecting hundreds of millions of people worldwide [52]. In recent years, ketamine has been shown to have unique rapid neuroprotective and antidepressant therapeutic effects by acting through the glutamate system [53,54,55]. As a result, over the past few years, a growing body of evidence has supported the role of the excitatory amino acid neurotransmitter glutamate in the treatment of depression [56, 57]. Glutamate is one of the most important neurotransmitters in the body and plays an important role in neuronal excitation [58, 59]. This neuronal excitation is transient and is followed by inhibition. Excitotoxicity is a pathological process in which nerve cells are damaged and die as a result of overstimulation by neurotransmitters such as glutamate and similar substances [60, 61]. This excitotoxic neuronal death is closely associated with MDD [62, 63]. Inhibition of glutamate excitotoxicity improves depressive symptoms [57, 64]. As a result of the long-term effects of glutamate excitotoxicity, neurons in the mPFC brain region were on the verge of death, and neuronal mEPSC frequencies were significantly reduced, which is consistent with our previous findings [65,66,67]. We found that a subdose of perampanel, an AMPAR antagonist used clinically for the treatment of epilepsy, reduces the effects of glutamate excitotoxicity by decreasing the expression of Grin2b thereby protecting neurons. The action of perampanel targets the glutamatergic system and opens up a new area for the development of drugs to meet the needs of patients with major depressive disorder as an alternative to ketamine.
Perampanel-induced reduction in Grin2b expression may be due to the fact that AMPAR-mediated depolarization of the postsynaptic membrane is necessary for the opening of the NMDAR [17, 26, 28]. And this effect precisely reduces the reduced excitatory synaptic transmission due to glutamatergic neuroexcitotoxicity induced by high Grin2b expression in the mPFC of CSDS depression susceptible mice. This is consistent with previous reports as overexpression of NMDAR causes glutamatergic neuroexcitotoxicity reduced excitatory synaptic transmission [68,69,70]. However, the inhibition of AMPAR by perampanel leads to defects in excitatory synaptic transmission in the mPFC of Naïve mice, which is also the reason why Naïve mice injected with perampanel produce rapid depressive effects. The disparate effects of perampanel on total GluA1 expression in Sus mice and Ctrl mice may be associated with variations in their initial GluN2B expression levels. Previous studies have established that an increase in postsynaptic AMPA receptor levels can retroactively enhance presynaptic glutamate release [31, 45,46,47,48,49]. We propose that the upregulation of postsynaptic Gria1, resulting from Grin2b reduction in Sus mice, underlies the rescue of mEPSC frequency. However, this elevated Gria1 expression did not lead to an increase in mEPSC amplitude, an effect that was abolished by the inhibitory action of perampanel on AMPA receptor function [20,21,22, 24]. The observed reduction in mEPSC amplitude in the NP0.5 group further corroborates this interpretation.
Non-competitive open channel NMDA receptor antagonists, including phencyclidine (PCP) and ketamine, may produce psychotomimetic effects when used acutely [71,72,73]. Thus, the clinical applicability of noncompetitive open-channel NMDA receptor antagonists (e.g., ketamine) appears to be limited by their propensity to elicit psychotomimetic effects. NMDAR are tetrameric proteins composed of two Grin1 subunits and two Grin2 subunits. NMDA receptors containing the Grin2b subunits are predominantly localized in the forebrain, including the mPFC and the hippocampus, a region implicated in the pathophysiology of major depressive disorder [29, 74]. Considering the acute psychomimetic side effects of ketamine, selective antagonists of the Grin2b subtype of the NMDA receptor appear to be of little risk. Indeed, the Grin2b antagonist Ro 25-6981 was shown to have antidepressant-like properties in a forced swimming trial [75]. A recent double-blind, randomized, placebo-controlled study demonstrated significant antidepressant effects of the selective Grin2b antagonist CP-101,606 (traxoprodil) in patients with refractory major depression [76]. The antidepressant effect of perampanel by reducing Grin2b expression may account for its lack of addiction and hallucinations.
Previous studies have demonstrated the rapid antidepressant effects of 0.5 mg/kg of ketamine, particularly in refractory depression [77, 78]. The first Randomized Controlled Trial (RCT) study used a dose of 0.5 mg/kg, which has been used in subsequent studies and is currently the most commonly used therapeutic dose in clinical practice [79, 80]. This is why we chose 0.5 mg/kg perampanel. In addition, we did not perform sucrose preference testing during depression-related behavioral testing because of its unsuitability for application in detecting rapid depressive and antidepressant effects.
Taken together, our research indicates that perampanel may be another potential antidepressant targeting the neuronal glutamate system, exhibiting rapid antidepressant effects. These findings suggest that perampanel could represent a potential precision therapy for epilepsy comorbid with depression. However, validating this definitively necessitates further investigation in a validated comorbid model.
Data availability
The mRNA-seq data generated in this study have been deposited in the Genome Sequence Archive under accession code CRA020238 (https://ngdc.cncb.ac.cn/gsa). All requests for raw data may be sent to the corresponding author.
References
The burden of mental disorders across the states of India: the Global Burden of Disease Study 1990–2017. Lancet Psychiatry, 2020. 7:148-61.
Hoppen TH, Priebe S, Vetter I, Morina N. Global burden of post-traumatic stress disorder and major depression in countries affected by war between 1989 and 2019: a systematic review and meta-analysis. BMJ Glob Health. 2021;6:e006303.
Malhi GS, Mann JJ. Depression. Lancet. 2018;392:2299–312.
Berton O, Nestler EJ. New approaches to antidepressant drug discovery: beyond monoamines. Nat Rev Neurosci. 2006;7:137–51.
Mann JJ. The medical management of depression. N Engl J Med. 2005;353:1819–34.
Krishnan V, Nestler EJ. The molecular neurobiology of depression. Nature. 2008;455:894–902.
Gewin V. Mental health: under a cloud. Nature. 2012;490:299–301.
McIntyre RS, Rosenblat JD, Nemeroff CB, Sanacora G, Murrough JW, Berk M, et al. Synthesizing the evidence for ketamine and esketamine in treatment-resistant depression: an international expert opinion on the available evidence and implementation. Am J Psychiatry. 2021;178:383–99.
Brendle M, Ahuja S, Valle MD, Moore C, Thielking P, Malone DC, et al. Safety and effectiveness of intranasal esketamine for treatment-resistant depression: a real-world retrospective study. J Comp Eff Res. 2022;11:1323–36.
Fedgchin M, Trivedi M, Daly EJ, Melkote R, Lane R, Lim P, et al. Efficacy and safety of fixed-dose esketamine nasal spray combined with a new oral antidepressant in treatment-resistant depression: results of a randomized, double-blind, active-controlled study (TRANSFORM-1). Int J Neuropsychopharmacol. 2019;22:616–30.
Nowacka A, Borczyk M. Ketamine applications beyond anesthesia - a literature review. Eur J Pharmacol. 2019;860:172547.
Ivan Ezquerra-Romano I, Lawn W, Krupitsky E, Morgan CJA. Ketamine for the treatment of addiction: evidence and potential mechanisms. Neuropharmacology. 2018;142:72–82.
Varì MR, Ricci G, Cavallo M, Pichini S, Sirignano A, Graziano S. Ketamine: from prescription anaesthetic to a new psychoactive substance. Curr Pharm Des. 2022;28:1213–20.
Zanos P, Gould TD. Mechanisms of ketamine action as an antidepressant. Mol Psychiatry. 2018;23:801–11.
Zanos P, Moaddel R, Morris PJ, Georgiou P, Fischell J, Elmer GI, et al. NMDAR inhibition-independent antidepressant actions of ketamine metabolites. Nature. 2016;533:481–6.
Suzuki K, Nosyreva E, Hunt KW, Kavalali ET, Monteggia LM. Effects of a ketamine metabolite on synaptic NMDAR function. Nature. 2017;546:E1–e3.
Hansen KB, Yi F, Perszyk RE, Furukawa H, Wollmuth LP, Gibb AJ, et al. Structure, function, and allosteric modulation of NMDA receptors. Journal of General Physiology. 2018;150:1081–105.
Bernard R, Kerman IA, Thompson RC, Jones EG, Bunney WE, Barchas JD, et al. Altered expression of glutamate signaling, growth factor, and glia genes in the locus coeruleus of patients with major depression. Mol Psychiatry. 2011;16:634–46.
Gerhard DM, Pothula S, Liu RJ, Wu M, Li XY, Girgenti MJ, et al. GABA interneurons are the cellular trigger for ketamine’s rapid antidepressant actions. J Clin Invest. 2020;130:1336–49.
Franco V, Crema F, Iudice A, Zaccara G, Grillo E. Novel treatment options for epilepsy: focus on perampanel. Pharmacol Res. 2013;70:35–40.
Ceolin L, Bortolotto ZA, Bannister N, Collingridge GL, Lodge D, Volianskis A. A novel anti-epileptic agent, perampanel, selectively inhibits AMPA receptor-mediated synaptic transmission in the hippocampus. Neurochem Int. 2012;61:517–22.
Rogawski, MA and T Hanada, Preclinical pharmacology of perampanel, a selective non-competitive AMPA receptor antagonist. Acta Neurol Scand Suppl 2013:19-24. https://doi.org/10.1111/ane.12100.
Catena-Dell’Osso M, Caserta A, Baroni S, Nisita C, Marazziti D. The relationship between epilepsy and depression: an update. Curr Med Chem. 2013;20:2861–7.
Wang T, Wang L, Li L, Ma L, Liu X. Effects of perampanel on cognitive behavior and GluR1 expression in immature mice of temporal lobe epilepsy. Biochem Biophys Res Commun. 2022;588:68–74.
Beaurain M, Salabert AS, Payoux P, Gras E, Talmont F. NMDA receptors: distribution, role, and insights into neuropsychiatric disorders. Pharmaceuticals (Basel). 2024;17:1265.
Shinohara R, Aghajanian GK, Abdallah CG. Neurobiology of the rapid-acting antidepressant effects of ketamine: impact and opportunities. Biol Psychiatry. 2021;90:85–95.
Raab-Graham KF, Workman ER, Namjoshi S, Niere F. Pushing the threshold: How NMDAR antagonists induce homeostasis through protein synthesis to remedy depression. Brain Res. 2016;1647:94–104.
Paoletti P, Bellone C, Zhou Q. NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. Nature Reviews Neuroscience. 2013;14:383–400.
Li N, Lee B, Liu R-J, Banasr M, Dwyer JM, Iwata M, et al. mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists. Science. 2010;329:959–64.
Dong WT, Long LH, Deng Q, Liu D, Wang JL, Wang F, et al. Mitochondrial fission drives neuronal metabolic burden to promote stress susceptibility in male mice. Nat Metab. 2023;5:2220–36.
Shepherd JD, Huganir RL. The cell biology of synaptic plasticity: AMPA receptor trafficking. Annu. Rev. Cell Dev. Biol. 2007;23:613–43.
Lussier MP, Sanz-Clemente A, Roche KW. Dynamic regulation of N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by posttranslational modifications. Journal of Biological Chemistry. 2015;290:28596–603.
Carvalho AL, Duarte CB, Carvalho AP. Regulation of AMPA receptors by phosphorylation. Neurochemical research. 2000;25:1245–55.
Cao Y-Y, Wu L-L, Li X-N, Yuan Y-L, Zhao W-W, Qi J-X, et al. Molecular mechanisms of AMPA receptor trafficking in the nervous system. International Journal of Molecular Sciences. 2023;25:111.
Widagdo J, Guntupalli S, Jang SE, Anggono V. Regulation of AMPA receptor trafficking by protein ubiquitination. Frontiers in Molecular Neuroscience. 2017;10:347.
Grados M, Salehi M, Lotfi A, Dua S, Xie I. A selective review of inhibitors of protein kinase C gamma: a neuroplasticity-related common pathway for psychiatric illness. Frontiers in Drug Delivery. 2024;4:1364037.
Song I, Huganir RL. Regulation of AMPA receptors during synaptic plasticity. Trends in neurosciences. 2002;25:578–88.
Gao Z, Van Beugen BJ, De Zeeuw CI. Distributed synergistic plasticity and cerebellar learning. Nature Reviews Neuroscience. 2012;13:619–35.
Zhu J, Lee KY, Jewett KA, Man H-Y, Chung HJ, Tsai N-P. Epilepsy-associated gene Nedd4-2 mediates neuronal activity and seizure susceptibility through AMPA receptors. PLoS genetics. 2017;13:e1006634.
Haouari S, Vourc’h P, Jeanne M, Marouillat S, Veyrat-Durebex C, Lanznaster D, et al. The roles of NEDD4 subfamily of HECT E3 ubiquitin ligases in neurodevelopment and neurodegeneration. International journal of molecular sciences. 2022;23:3882.
Donovan P, Poronnik P. Nedd4 and Nedd4-2: ubiquitin ligases at work in the neuron. The international journal of biochemistry & cell biology. 2013;45:706–10.
Béïque J-C, Lin D-T, Kang M-G, Aizawa H, Takamiya K, Huganir RL. Synapse-specific regulation of AMPA receptor function by PSD-95. Proceedings of the National Academy of Sciences. 2006;103:19535–40.
Ehrlich I, Malinow R. Postsynaptic density 95 controls AMPA receptor incorporation during long-term potentiation and experience-driven synaptic plasticity. Journal of Neuroscience. 2004;24:916–27.
Bhattacharyya S, Biou V, Xu W, Schlüter O, Malenka RC. A critical role for PSD-95/AKAP interactions in endocytosis of synaptic AMPA receptors. Nature neuroscience. 2009;12:172–81.
Sanchez-Prieto J, Budd DC, Herrero I, Vázquez E, Nicholls DG. Presynaptic receptors and the control of glutamate exocytosis. Trends in neurosciences. 1996;19:235–9.
Chater TE, Goda Y. The role of AMPA receptors in postsynaptic mechanisms of synaptic plasticity. Frontiers in cellular neuroscience. 2014;8:401.
Garthwaite J, Boulton C. Nitric oxide signaling in the central nervous system. Annual review of physiology. 1995;57:683–706.
Arancio O, Kiebler M, Lee CJ, Lev-Ram V, Tsien RY, Kandel ER, et al. Nitric oxide acts directly in the presynaptic neuron to produce long-term potentiationin cultured hippocampal neurons. Cell. 1996;87:1025–35.
Regehr WG, Carey MR, Best AR. Activity-dependent regulation of synapses by retrograde messengers. Neuron. 2009;63:154–70.
Patsalos PN. The clinical pharmacology profile of the new antiepileptic drug perampanel: a novel noncompetitive AMPA receptor antagonist. Epilepsia. 2015;56:12–27.
Schulze-Bonhage A. Perampanel for epilepsy with partial-onset seizures: a pharmacokinetic and pharmacodynamic evaluation. Expert Opinion on Drug Metabolism & Toxicology. 2015;11:1329–37.
Marx W, Penninx B, Solmi M, Furukawa TA, Firth J, Carvalho AF, et al. Major depressive disorder. Nat Rev Dis Primers. 2023;9:44.
Machado-Vieira R, Salvadore G, Diazgranados N, Zarate CA Jr. Ketamine and the next generation of antidepressants with a rapid onset of action. Pharmacol Ther. 2009;123:143–50.
Murrough JW. Ketamine as a novel antidepressant: from synapse to behavior. Clin Pharmacol Ther. 2012;91:303–9.
Caddy C, Giaroli G, White TP, Shergill SS, Tracy DK. Ketamine as the prototype glutamatergic antidepressant: pharmacodynamic actions, and a systematic review and meta-analysis of efficacy. Ther Adv Psychopharmacol. 2014;4:75–99.
Stewart CA, Reid IC. Antidepressant mechanisms: functional and molecular correlates of excitatory amino acid neurotransmission. Mol Psychiatry. 2002;7:S15–22.
Kim J, Kim TE, Lee SH, Koo JW. The role of glutamate underlying treatment-resistant depression. Clin Psychopharmacol Neurosci. 2023;21:429–46.
Zhou Y, Danbolt NC. Glutamate as a neurotransmitter in the healthy brain. J Neural Transm (Vienna). 2014;121:799–817.
Teleanu RI, Niculescu AG, Roza E, Vladâcenco O, Grumezescu AM, Teleanu DM. Neurotransmitters—key factors in neurological and neurodegenerative disorders of the central nervous system. International journal of molecular sciences. 2022;23:5954.
Mehta A, Prabhakar M, Kumar P, Deshmukh R, Sharma PL. Excitotoxicity: bridge to various triggers in neurodegenerative disorders. Eur J Pharmacol. 2013;698:6–18.
Wang Y, Qin ZH. Molecular and cellular mechanisms of excitotoxic neuronal death. Apoptosis. 2010;15:1382–402.
Sapolsky RM. The possibility of neurotoxicity in the hippocampus in major depression: a primer on neuron death. Biol Psychiatry. 2000;48:755–65.
Lee AL, Ogle WO, Sapolsky RM. Stress and depression: possible links to neuron death in the hippocampus. Bipolar Disord. 2002;4:117–28.
Niciu MJ, Ionescu DF, Richards EM, Zarate CA. Jr., Glutamate and its receptors in the pathophysiology and treatment of major depressive disorder. J Neural Transm (Vienna). 2014;121:907–24.
Fan J, Guo F, Mo R, Chen LY, Mo JW, Lu CL, et al. O-GlcNAc transferase in astrocytes modulates depression-related stress susceptibility through glutamatergic synaptic transmission. J Clin Invest. 2023;133:e160016.
Guo F, Fan J, Liu JM, Kong PL, Ren J, Mo JW, et al. Astrocytic ALKBH5 in stress response contributes to depressive-like behaviors in mice. Nat Commun. 2024;15:4347.
Kuang XJ, Zhang CY, Yan BY, Cai WZ, Lu CL, Xie LJ, et al. P2X2 receptors in pyramidal neurons are critical for regulating vulnerability to chronic stress. Theranostics. 2022;12:3703–18.
Rodriguez-Chavez V, Moran J, Molina-Salinas G, Zepeda Ruiz WA, Rodriguez MC, Picazo O, et al. Participation of Glutamatergic Ionotropic Receptors in Excitotoxicity: The Neuroprotective Role of Prolactin. Neuroscience. 2021;461:180–93.
Urushitani M, Shimohama S, Kihara T, Sawada H, Akaike A, Ibi M, et al. Mechanism of selective motor neuronal death after exposure of spinal cord to glutamate: involvement of glutamate-induced nitric oxide in motor neuron toxicity and nonmotor neuron protection. Ann Neurol. 1998;44:796–807.
Fan G, Liu M, Liu J, Huang Y. The initiator of neuroexcitotoxicity and ferroptosis in ischemic stroke: Glutamate accumulation. Front Mol Neurosci. 2023;16:1113081.
Nussenzveig IZ, Sircar R, Wong ML, Frusciante MJ, Javitt DC, Zukin SR. Polyamine effects upon N-methyl-D-aspartate receptor functioning: differential alteration by glutamate and glycine site antagonists. Brain Res. 1991;561:285–91.
Krystal JH, Karper LP, Seibyl JP, Freeman GK, Delaney R, Bremner JD, et al. Subanesthetic effects of the noncompetitive NMDA antagonist, ketamine, in humans. Psychotomimetic, perceptual, cognitive, and neuroendocrine responses. Arch Gen Psychiatry. 1994;51:199–214.
Krystal JH, Abi-Saab W, Perry E, D’Souza DC, Liu N, Gueorguieva R, et al. Preliminary evidence of attenuation of the disruptive effects of the NMDA glutamate receptor antagonist, ketamine, on working memory by pretreatment with the group II metabotropic glutamate receptor agonist, LY354740, in healthy human subjects. Psychopharmacology (Berl). 2005;179:303–9.
Liu W, Ge T, Leng Y, Pan Z, Fan J, Yang W, et al. The role of neural plasticity in depression: from hippocampus to prefrontal cortex. Neural Plast. 2017;2017:6871089.
Maeng S, Zarate CA Jr., Du J, Schloesser RJ, McCammon J, Chen G, et al. Cellular mechanisms underlying the antidepressant effects of ketamine: role of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors. Biol Psychiatry. 2008;63:349–52.
Preskorn SH, Baker B, Kolluri S, Menniti FS, Krams M, Landen JW. An innovative design to establish proof of concept of the antidepressant effects of the NR2B subunit selective N-methyl-D-aspartate antagonist, CP-101,606, in patients with treatment-refractory major depressive disorder. J Clin Psychopharmacol. 2008;28:631–7.
Kohtala S. Ketamine-50 years in use: from anesthesia to rapid antidepressant effects and neurobiological mechanisms. Pharmacol Rep. 2021;73:323–45.
Shiroma PR, Thuras P, Wels J, Albott CS, Erbes C, Tye S, et al. A randomized, double-blind, active placebo-controlled study of efficacy, safety, and durability of repeated vs single subanesthetic ketamine for treatment-resistant depression. Transl Psychiatry. 2020;10:206.
McGirr A, Berlim MT, Bond DJ, Fleck MP, Yatham LN, Lam RW. A systematic review and meta-analysis of randomized, double-blind, placebo-controlled trials of ketamine in the rapid treatment of major depressive episodes. Psychol Med. 2015;45:693–704.
Murrough JW, Iosifescu DV, Chang LC, Al Jurdi RK, Green CE, Perez AM, et al. Antidepressant efficacy of ketamine in treatment-resistant major depression: a two-site randomized controlled trial. Am J Psychiatry. 2013;170:1134–42.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. 32271062), STI2030-Major Projects 2022ZD0204700, Guangzhou Key Research Program on Brain Science (202206060001); Guangdong-Hong Kong Joint Laboratory for Psychiatric Disorders (2023B1212120004). The China Postdoctoral Science Foundation, No.76 General Fund, 2024, (K125280101).
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X. Cao, W. Wu, X. Tan and J. Liu conceived and designed the study. J. Liu, Y. Zhang, F. Guo, B. Li performed the experiments and analyzed the data. H. Chen, Y. Mo, J. Kong participate in the experiments. J. Liu, X. Cao wrote the manuscript. All authors provided the inputs and reviewed the manuscript.
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Liu, JM., Zhang, YL., Guo, F. et al. Treatment with perampanel alleviates depression-like behavior in mice via modulating GluN2B expression to improve excitatory synaptic transmission. Transl Psychiatry 16, 90 (2026). https://doi.org/10.1038/s41398-026-03874-1
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DOI: https://doi.org/10.1038/s41398-026-03874-1
