TRPM2 regulates the excitability of hippocampal neurons through Kv7 channel in epilepsy

doi:10.21203/rs.3.rs-1498239/v1

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TRPM2 regulates the excitability of hippocampal neurons through Kv7 channel in epilepsy

https://doi.org/10.21203/rs.3.rs-1498239/v1

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Transient receptor potential Melastain2 (TRPM2) ion channel is a non-selective cationic channel that can permeate Ca²⁺ and is highly expressed in the brain, especially in the cerebral cortex, hippocampus, and striatum, which are critical for cognitive function. Current research evidence indicates that TRPM2 is closely associated with various neurological diseases such as epilepsy. In this study, TRPM2 gene knockout mice were used to study the effect of TRPM2 on the susceptibility to epilepsy in mice and the specific mechanism of the impact of TRPM2 on epilepsy by affecting neuronal excitability. In PTZ chronic kindling epilepsy model, we have found that TRPM2 knockout mice were more susceptible to epilepsy than WT mice. Furthermore, the neuronal excitability in the hippocampal CA1 region of TRPM2 knockout mice is significantly increased. Compared with WT group, there were no significant differences in the input resistance and afterhyperpolarization of CA1 neurons in TRPM2 knockout mice. Firing adaptation rate of hippocampal CA1 pyramidal neurons of TRPM2 knockout mice is lower than that of WT mice. We found that activation of Kv7 channel by retigabine reduced the firing frequency of action potential in the hippocampal pyramidal neurons of TRPM2 knockout mice. However, inhibiting Kv7 channel increased the firing frequency of action potential in hippocampal pyramidal neurons of WT mice. In animal models, we confirmed that activation of Kv7 channel can effectively reduce epileptic seizures in TRPM2 knockout mice. These results suggest that hippocampal CA1 pyramidal neurons in TRPM2 knockout mice may increase neuronal excitability by inhibiting the Kv7 channel, thus affecting the susceptibility to epilepsy. This study carefully explores the reasons why TRPM2 knockout is more susceptible to epilepsy, which is helpful to understand the occurrence and development of epilepsy, and provides new ideas for the treatment technology and methods of epilepsy.

Epilepsy

TRPM2

Hippocampus CA1 region

M current

Neuronal excitability

Action potential

Epilepsy is a common chronic brain disease that affects approximately 70 million people worldwide, making it one of the most common neurological disorders [1]. The incidence, prevalence, and mortality of epilepsy vary across countries with different economies [2]. Epidemiology shows that the overall prevalence of epilepsy is 3.6‰ to 7.0‰, of which 60% originated in childhood, and the incidence of epilepsy in children is 10–15 times higher than that in adults. Epilepsy is considered an acute transient complex neurobehavioral disease [3]. The disease is characterized by recurrent seizures, which are due to aberrant neuronal networks resulting in synchronous discharges. Increased excitability of neurons in various brain regions plays an important role in the etiology of epilepsy. Beyond debilitating seizures, comorbidities of this epileptic disorder are more common, such as anxiety, cognitive impairment, depression, sleep disorders and so on. There are many treatments for epilepsy, including antiseizure medication, ketogenic diet, epilepsy surgery and neuromodulation techniques such as responsive neural stimulation. Antiseizure medication might suppress seizures in up to two-thirds of all individuals but do not alter long-term prognosis [4]. Although numerous studies have been conducted on the etiology of epilepsy, clear cellular and molecular mechanisms involved in the etiology of epilepsy are still unclear. Therefore, it is rather urgent to find out underlying causes and develop more effective treatments to improve epilepsy patients’ quality of life.

The pathogenesis of epilepsy is complicated, which involve ion channel- and synapto-pathologies, aberrant gene expression, neuronal network and blood-brain barrier dysfunction as well as inflammation, innate and adaptive immunity-mediated damage, and so on [5]. About 25% of genes identified in epilepsy encode ion channels [6]. The functions of ion channels include controlling ion flow across the membrane, establishing action potential, maintaining intracellular ion balance and regulating cell volume. The association between ion channels and epilepsy may help to reveal the potential mechanism of epilepsy [7]. There are a variety of ion channels involved in the development of epilepsy, such as voltage-gated ion channels, including sodium channels (SCN1A [8], SCN1B [9], SCN2A [10, 11], SCN8A [12, 13]), potassium channel (KCNA1 [14, 15], KCNA2 [16, 17], KCNB1, KCNC1, KCNMA1 [18, 19], KCNQ2/3 [20–22], KCNT1, KCTD7), calcium channel (CACNA1A [23, 24]), hyperpolarization-activated cyclic nucleotide-gated channels (HCN channels [25, 26]), and ligand gated ion channel, including NMDAR, GABAAR, nAChR, etc. [6]. These ion channels affect the occurrence of epilepsy in various ways, but the mechanisms of these various ion channels in epilepsy have not yet been fully elucidated. Therefore, it is urgent for us to explore the exact pathogenesis of epilepsy to find more effective treatment methods.

Transient receptor potential melastain2 (TRPM2) ion channel, which belongs to the transient receptor potential (TRP) superfamily, is a non-selective, Ca²⁺ permeable cation channel. The activation of TRPM2 can cause influx of Ca²⁺ to the cytoplasm, and further lead to the production of multiple cytokines, cell migration, oxidative stress, inflammation, cell death and many other pathophysiological processes. Emerging evidence demonstrates that TRPM2 is closely related to neurological diseases, such as multiple sclerosis [27], epilepsy [28], cerebral ischemia-reperfusion injury [29], Alzheimer's disease [30], Parkinson's disease [31], neuropathic pain [32] and bipolar disorder [31], etc.. Li et al. [33] revealed that TRPM2 channel participated in the pathological process of Alzheimer's disease through Aβ₄₂-induced TRPM2 dependent positive feedback loop of hippocampal neurotoxicity in neurons, and through inflammatory response and oxidative stress in glial cells [34]. In ischemic brain injury, TRPM2 channel activation can inhibit GluN2A mediating survival signal pathway and enhance GluN2B mediating death signal pathway, resulting in neuronal death [29, 35–37]. Ye et al. [37] proposed that after ischemia, TRPM2 channel is activated, resulting in the increase of [Zn²⁺]_i, which acts on mitochondria, causes mitochondrial dysfunction and the production of mitochondrial ROS, and eventually leads to neuronal death. Studies have shown that TRPM2 knockout can alleviate the emotional and cognitive impairment of pilocarpine induced epilepsy model [28].What is more, TRPM2 affects JME phenotype by mediating the destructive effect of EFHC1 mutation in juvenile myoclonic epilepsy [38–40]. TRPM2 channel may participate in the pathophysiological process of Parkinson's disease by involved in the explosive discharge of GABAergic neurons in substantia nigra reticular induced by NMDA [41] and the inhibition of TRPM2 may mediate Akt/GSK-3 β/Capsase-3 signaling pathway providing neuroprotective effect on PD model [42]. This suggests that TRPM2 may be an important target for the treatment of related neurological diseases.

Potassium (K⁺) channels, being the most diverse of all ion channels, underlie a robust number of functions controlling the excitability of neurons. Neuronal KCNQ-encoded voltage-dependent K⁺ channels (Kv7) subunits are widely expressed in the central nervous system, where its steady outwardly rectifying current functions as “brakes” for neurons receiving persistent excitatory input. It is becoming increasingly evident that Kv7 channels can be valid targets for epilepsy. The common denominator is probably neuronal hyperexcitability in different brain areas, which can be successfully attenuated by pharmacological increment of Kv7 channel activity [43]. Lipinsky et al. found that removal of external Ca²⁺ in wild-type Kv7.1 channels produced a large, voltage-dependent inactivation, which differed from N- or C-type mechanisms [44]. TRPM2 ion channel is permeable to various cations, such as Na⁺, K⁺, Ca²⁺, and involved in the development of various neurological diseases. Epilepsy is also associated with a variety of ions, such as Na⁺, K⁺, and Ca²⁺. Though there are some researches on the effects of TRPM2 on seizures, studies on specific mechanisms of TRPM2 channels in epilepsy are few, let alone whether there is a connection between TRPM2 and Kv7 channel.

In summary, TRPM2 and Kv7 channel activity are closely related to epilepsy, but the specific relationship and the molecular mechanisms involved in epilepsy are not clear. Therefore, this study intends to establish a chronic kindling epilepsy model through TRPM2 knockout mice to explore the specific mechanism of TRPM2 on regulating the excitability of hippocampal neurons through the Kv7 channel using electrophysiological techniques to provide new ideas and inspiration for the development of epilepsy drugs and clinical treatment.

Animals

TRPM2-KO (TRPM2^−/−) mice were obtained from the Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Japan, and they (backcrossed for 12 generations onto the C57BL/6 background) were maintained in our laboratory. C57BL/6 mice (male, 25–30g) were also used in this study. The mice used in our study were littermates, which were bred in the Zhejiang University laboratory animal center. They were group-housed 4–6 mice per cage with free access to food and water and kept at a constant ambient temperature of 24 ± 2°C, with 40–60% humidity, under a 12 h light/dark cycle. Genomic DNA was extracted from the toes of 4-week-old male mice for TRPM2^−/− mice genotype identification. The Primer sequences of PCR: Forward, 5’-CTT GGG TTG CAG TCA TAT GCA GGC-3’; Reverse, 5’-GCC CTC ACC ATC CGC TTC ACG ATG-3’. All experiments were performed in accordance with the requirements of Animal Experiment Ethics Committee of Zhejiang University. Efforts were made to minimize suffering and unnecessary use of animals.

Pentylenetetrazol treatment

C57BL/6 (WT) or TRPM2-KO male mice aged 6–8 weeks were intraperitoneally injected with 30 mg/kg PTZ solution at 10 ml/kg for 14 consecutive days. The behaviors of each mouse were observed for 30 min after each injection. Seizure grades were recorded according to Racine score. Mice were considered successfully when they had reached Racine Stage III or higher for three consecutive days.

Brain slice preparation

Briefly, mice (6-8-week-old) were anesthetized with isoflurane and immediately decapitated. Their brains were extracted and rapidly transferred into ice-cold and oxygenated(95% O₂ / 5%CO₂)cutting solutions (in mM: 234 Sucrose, 5 KCl, 1.25 NaH₂PO₄, 5 MgSO₄, 26 NaHCO₃, 25 D-glucose, 1 CaCl₂, PH 7.4, 300 mOsm). Transverse slices of hippocampus (300 µm for whole-cell recording) were cut with a tissue slicer (VT 1200S, Leica). The selected slices were transferred into oxygenated artificial cerebrospinal fluid (ACSF) (in mM: 124 NaCl, 2 KCl, 1.25 KH₂PO₄, 2 MgSO₄, 26 NaHCO₃, 10 D-glucose, 2 CaCl₂, PH 7.4, 300mOsm), and incubated in 32℃ water baths for 25 min, and then in room temperature (25℃) for at least an hour before further electrophysiological recording.

Electrophysiology recordings of brain slices

Whole-cell recordings were performed as described in articles [45, 46]. Hippocampal slices were transferred to the recording chamber at room temperature and perfused continuously with ACSF bubbled with 95% O₂ / 5% CO₂ to ensure adequate oxygenation of slices. CA1 pyramidal neurons were identified under infrared differential interference contrast (IR-DIC) optics based on their location and morphology. Borosilicate glass (Sutter instruments) pipettes (3–5 MΩ) were pulled with a horizontal pipette puller (P97, Sutter instruments) and were filled with artificial intracellular fluid. Pipettes were connected to the headstage of a Heka EPC 10 amplifier (Heka Elektronik), and fast and slow capacitances, as well as series resistance compensations, were carefully adjusted. Liquid junction potentials were not corrected. Series resistance was normally less than 20 MΩ and recordings exceeding 20% change in series resistance were terminated and discarded. Recordings were filtered at 2.0 kHz and digitized at 10 kHz.

To measure the action potentials, glass pipettes were filled with an intracellular solution, containing (in mM) 120 K-gluconate, 15KCl, 10 HEPES, 4 Mg-ATP, 0.3 Tris-GTP, and 0.5 EGTA (pH 7.3). APs were triggered by injecting currents, ranging from – 150 to + 290 pA, in increments of 10 pA steps in current-clamp mode. The number of spikes that were evoked by the injected currents in the hippocampal neurons isolated from WT or TRPM2-KO mice was compared. To measure spontaneous excitatory postsynaptic currents (sEPSC), glass pipettes were filled with another solution, containing (in mM) 100 CsCH₃SO₃, 20 KCl, 10 HEPES, 4 Mg-ATP, 0.3 Tris-GTP, 7 Tris2-phosphocreatine, and 3 Qx-314 (pH 7.3). The membrane potential was held at − 70 mV under voltage-clamp when spontaneous EPSCs were recorded. Igor pro was used to analyze action potentials, and Mini Analysis was used for sEPSCs analysis.

In the whole-cell recording, the cell membrane was applied with a series of stimulation pulses (typically hyperpolarized), and the transmembrane current was measured and recorded without ion channels being open. Input resistance was calculated according to Ohm's law. Change to current-clamp immediately after breaking the membrane, and then change the current to 0 pA and check the voltage value — the voltage value when I = 0 is resting membrane potential. We measured the mAHP peak voltages after inject current stimulation of the neuron. The difference value from the baseline is the amplitude of mAHP.

The method of recording Kv7 current(M current)was acquired from the reported literature[47, 48]. Briefly, pipettes were filled with an internal solution containing (in mM) 120 K-gluconate, 15KCl, 10 HEPES, 4 Mg-ATP, 0.3 Tris-GTP, and 0.5 EGTA (pH 7.3). The external ACSF was supplemented with tetrodotoxin (0.5 µM) and DNQX (10 µM) to block sodium current and non-NMDA receptor-mediated responses, respectively. The membrane potential was held at -20 mV for M-current activation. A deactivation protocol was applied. M current amplitude was measured as the relaxation current in response to a voltage step protocol from − 20 mV to return potentials of − 50 mV.

Statistical Analysis

All of the results are shown as means ± SEMs. The unpaired two-tailed Student’s t test was used to determine the statistical differences between the two means. A one-way or two-way analysis of variance (ANOVA) with Tukey’s post hoc analysis was used to conduct multiple comparisons of the means. Mann-Whitney or Kruskal-Wallis analysis was used to conduct multiple comparisons of the means with small number of experiments (n ≤ 6). Statistical significance was accepted for p value of < 0.05. p-value less than 0.05 was considered to be statistically significant, defined as *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. Calculation was performed in GraphPad Prism TM 8.0 (version 8.0; GraphPad Software Inc., USA) and SPSS 22.0 (IBM.Corp., USA).

TRPM2 knockout is more susceptible to seizures in PTZ chronic kindling epilepsy model

To investigate the effect of TRPM2 ion channel on epilepsy, PTZ chronic kindling epilepsy model was established by intraperitoneal injection of 30 mg/kg PTZ solution in TRPM2 gene knockout (TRPM2-KO) mice and C57BL/6 wild type (WT) mice for 14 consecutive days. Compared with WT group, the time to reach Racine Stage III was shorter in TRPM2-KO group, and the Racine Scores were significantly higher at the same time (3.40 ± 0.29 vs 1.96 ± 0.15, P < 0.001) (Fig. 1A). However, the duration of each seizure in WT group was significantly higher than that in TRPM2-KO group (37.45 ± 6.579s vs 22.49 ± 1.451s, P < 0.001) (Fig. 1C), and there was no significant difference in the latency to reach Racine stage 3 between these two groups (6.333 ± 1.520 days vs 4.565 ± 0.525 days, P > 0.05) (Fig. 1B). Compared with WT group, the kindling rate of TRPM2-KO group was significantly higher (79.31% vs 13.79%, P < 0.001) (Fig. 1D). And there was a significant increase in mortality of TRPM2-KO group (58.62% vs 13.79%, P < 0.001) (Fig. 1E). Combined with these results, we inferred that TRPM2-KO mice were more susceptible to epilepsy than WT mice.

The susceptibility of TRPM2-KO to epilepsy was due to increased firing frequency of APs instead of sEPSC

To explore why TRPM2-KO mice were more susceptible to epilepsy than WT mice, we recorded spontaneous excitatory postsynaptic current (sEPSC) of hippocampal CA1 pyramid neurons in both groups by whole-cell voltage-clamp. Amplitude and frequency of sEPSC in TRPM2-KO mice and WT mice were analyzed and compared (Fig. 2A). There was no significantly difference between sEPSC amplitude in TRPM2-KO group and that in WT group (TRPM2-KO -11.810 ± 1.659 pA vs WT -8.448 ± 0.596 pA, P > 0.05) (Fig. 2B), as well as frequency (TRPM2-KO 3.198 ± 0.393Hz vs WT 3.411 ± 0.380Hz, P > 0.05) (Fig. 2C). These results suggest that although TRPM2-KO was more susceptible to epilepsy than mice in the WT group, this outcome was not due to synaptic excitability during synaptic transmission.

To further explore the reason why TRPM2-KO mice are more susceptible to epilepsy, we recorded action potentials in hippocampal CA1 pyramidal neurons of TRPM2-KO group and WT group by current clamp (Fig. 2D), and then analyzed and compared the firing frequencies of action potentials. The results showed that the firing frequency of action potentials in hippocampal CA1 pyramidal neurons in TRPM2-KO group was significantly higher than that in WT group (P < 0.001) (Fig. 2E). This indicated that the excitability of hippocampal CA1 pyramidal neurons in TRPM2-KO group was higher than that in WT group.

To further verify the reliability of this result, we used TRPM2 inhibitor (2-APB, 100µmol/L for 1min) on WT and TRPM2-KO neurons. 2-APB is recognized to strongly inhibit TRPM2. Results showed that numbers of action potential in WT neurons were significantly increased after 2-APB (Fig. 2F, G), while TRPM2-KO neurons showed no differences (Fig. 2H, I), which was consistent with the previous results of TRPM2-KO. Our data show that 2-APB has no effect in slices from TRPM2 KO and support that 2-APB can mimic the phenotype observed in TRPM2-KO. Therefore, we inferred the susceptibility of TRPM2-KO to epilepsy was due to increased firing frequency of action potential (APs) instead of sEPSC.

Input resistance and mAHP were not reasons of higher excitability of TRPM2-KO neurons

To understand the causes of increased excitability of TRPM2-KO neurons, we examined several factors affecting the excitability of neurons respectively. Input resistance is an important factor affecting the excitability of neurons. Therefore, we calculated the input resistance of hippocampal pyramidal neurons in TRPM2-KO and WT, and conducted statistical analyzed. We found that there was no significant difference in input resistance between TRPM2-KO group and WT group (TRPM2-KO 204.80 ± 13.80 MΩ vs WT 195.40 ± 14.32 MΩ, P > 0.05) (Fig. 3A). Besides, resting membrane potentials between these two groups were not significantly different (TRPM2-KO -60.62 ± 0.37 mV vs WT -60.94 ± 0.31 mV, P > 0.05) (Fig. 3B). These results suggest that the increased excitability of hippocampal CA1 pyramidal neurons in TRPM2-KO is not the result of input resistance changes.

What’s more, based on known studies, we hypothesized that the alteration of excitability of TRPM2-KO neurons might be related to small-conductance Ca²⁺-activated K⁺ (SK) channel. To this end, we got weighted average the action potential traces of I = 290pA, and obtained the typical statistical trace of action potential at 290pA (Fig. 3C). The results showed that there was no significant difference in medium afterhyperpolarization (mAHP) between TRPM2-KO group and WT group when the injective current was I = 290 pA (Fig. 3D).

The adaption rate of hippocampal CA1 pyramidal neurons in TRPM2-KO was lower

The fitting curve showed that firing frequency would be slower during the repeated firing process and the firing frequency of TRPM2-KO neurons decreased even more slowly than that of WT (Fig. 3E). To further compare the difference of discharge adaptation between neurons of TRPM2-KO mice and WT mice, discharge adaptation was calculated and analyzed. The discharge adaptation is calculated as follows: discharge fitness = (F₀-F_SS)/F₀, where F₀ is the initial discharge frequency and F_SS is the steady discharge frequency. According to statistics, the discharge adaptation rate of mouse neurons in WT group was higher than that in TRPM2-KO group (0.6174 ± 0.04105 vs 0.4908 ± 0.03538, P < 0.05) (Fig. 3F). This was consistent with the discharge diagram in Fig. 5A, which could be observed that hippocampal CA1 neurons had certain discharge adaptation. This indicated that compared with the TRPM2-KO group, CA1 neurons in WT group had more obvious discharge adaptation.

The increased excitability of TRPM2-KO neurons is related to the Kv7 channel

We hypothesize that CA1 neurons in TRPM2-KO may be more excitable due to reduced Kv7 function. To verify this hypothesis, we recorded Kv7 current directly to prove that TRPM2-KO have a deficit in Kv7 function. Results showed that after administration of XE991 (a Kv7 channel blocker), WT neurons had a significantly reduced Kv7 current, while the Kv7 current in TRPM2-KO neurons had no differences (Fig. 4A-D).

And then, we recorded action potentials before and after TRPM2-KO and WT brain slices were added with Retigabine (ethyl N-[2-amino-4- [(4-fluorophenyl) methylamino]phenyl] carbamate) (10µmol/L for 10min) or XE991 dihydrochloride (10, 10-bis (4-pyridinylmethyl) − 9(10H) - anthracenone dihydrochloride) (10µmol/L for 10min), respectively (Fig. 5A). Retigabine is a Kv7 channel agonist [49, 50] and XE991 is a Kv7 channel blocker [51, 52]. Our results showed that the firing frequency of TRPM2-KO neurons significantly decreased after the administration of retigabine (P < 0.001), while the administration of XE991 made no significant difference (Fig. 5B, C). After XE991 was added, the firing frequency of WT neurons increased significantly (P < 0.001), and retigabine made no sense (Fig. 5B, C). There was no significant difference between TRPM2-KO + Retigabine group and the WT group (P > 0.05) (Fig. 5B, C), as well as WT + XE991 group and the TRPM2-KO group (P > 0.05) (Fig. 5B, C). These results indicated that Kv7 activity was reduced in TRPM2-KO neurons, and XE991 reduced maximal effect in TRPM2-KO neurons, whereas retigabine in contrast had a more pronounced effect in TRPM2-KO.

Kv7 channel agonists can effectively reduce seizures in TRPM2-KO mice

Previous results have shown that the application of Kv7 channel agonists can effectively reduce the excitability of neurons in TRPM2-KO mice. In order to verify the effect of Kv7 channel agonists on epilepsy, the PTZ acute epilepsy model was used for animal verification due to the uncertainties of long-term and sustained administration in the chronic kindling model. Male C57BL/6 (WT) or TRPM2-KO mice were intraperitoneally injected 10 mg/kg Retigabine or 10 mg/kg XE991 in advance, and then 50 mg/kg PTZ was injected once after 15 min. After injection, the behavior of each mouse was observed for 30 min. Seizure grades were recorded according to Racine score (Fig. 6A). In PTZ acute epilepsy model, intraperitoneal injection of 10 mg/kg Retigabine significantly reduced seizure grade in TRPM2-KO mice (WT 3.80 ± 0.55; TRPM2-KO 4.10 ± 0.28; WT + XE991 5.00 ± 0.00; TRPM2-KO + Retigabine 1.50 ± 0.34; Among them, TRPM2-KO 4.10 ± 0.28 vs TRPM2-KO + Retigabine 1.50 ± 0.34, P < 0.001) (Fig. 6B). However, the latency of seizures in WT group was significantly shortened after intraperitoneal injection of 10 mg/kg XE991 (WT 231.90 ± 32.25 s; TRPM2-KO 242.8 ± 26.27 s; WT + XE991 127.20 ± 24.56s; TRPM2-KO + Retigabine mice had no grand mal seizure. WT 231.90 ± 32.25s vs WT + XE991 127.20 ± 24.56s, P < 0.05) (Fig. 6C), and the duration of seizures was significantly reduced (WT 149.20 ± 75.77s; TRPM2-KO 19.33 ± 0.55 s; WT + XE991 13.33 ± 1.12 s; TRPM2-KO + Retigabine had no grand mal seizure. WT 149.20 ± 75.77s vs WT + XE991 13.33 ± 1.12s, P < 0.001) (Fig. 6D).

In the PTZ acute epilepsy model, the incidence of seizures and mortality was 100% in the WT group after intraperitoneal injection of XE991, while there were almost no grand mal seizures or deaths in the TRPM2-KO group after intraperitoneal injection of Retigabine. Combined with these all results, it suggested that Retigabine could reduce seizures in TRPM2-KO mice to some extent.

Epilepsy is a common, debilitating neurological condition which is characterized by recurrent seizures. An epileptic seizure is an example of severely perturbed neural activity, which is accompanied by pronounced changes in intracellular and extracellular ion concentrations. These striking events often involve severe disruption of brain function due to increased excitation and synchronization in underlying brain networks. Changes in membrane voltage brought about by ion fluxes through voltage and transmitter-gated channels represent the basis of neural activity. The animal models of epilepsy include chemical models, physical models and genetic models. Common convulsants include pentylenetetrazol, kainic acid, pilocarpine, etc.. One of the most typical convulsants is PTZ, which can be used to establish animal models of both acute and chronic epilepsy. The PTZ chronic kindling model was first proposed by Mason and Cooper in 1972 [53]. In rodents, acute myoclonus and tonic dilation are caused by PTZ at a threshold dose, while the kindling phenomenon is caused by repeated administration at a subthreshold concentration. Recently, other researchers continuously improved the PTZ chronic kindling model. Studies have shown that PTZ-induced kindling is long-term progress, and once PTZ kindling epilepsy is successful, a solid epileptic response will occur when PTZ is reapplied several months later, even if no drugs are used to stimulate convulsions [54]. In our study, we repeated intraperitoneal injection of PTZ at a subthreshold dose (30 mg/kg) to get PTZ chronic kindling model. Our data showed that the kindling rate and the mortality were significantly increased in TRPM2 knockout mice. It suggested that TRPM2 channel may play an important role in the development of epilepsy.

TRPM2 channel has emerged as an important calcium (Ca²⁺) signalling mechanism in a variety of cells, contributing to cellular functions that include cell motility and cell death. TRPM2 is a non-selective, Ca²⁺-permeable cation channel, which can mobilize Ca²⁺ from both extracellular and intracellular compartments and as such its biological significance is related to cellular functions that are regulated by [Ca²⁺]i. TRPM2 is highly expressed in the brain, especially in the cerebral cortex [55], hippocampus [56, 33] and striatum [57], which are critical to cognitive function. It is highly sensitive to oxidative stress or reactive oxygen species (ROS), and can be used as a sensor for oxidative stress. TRPM2 has been associated with central nervous system (CNS) pathologies such as bipolar disorder, amyotrophic lateral sclerosis, parkinsonism–dementia and epilepsy and so on [27, 28, 58, 59]. Zhu et al. found that in the lipopolysaccharide-induced endotoxin sepsis model, TRPM2 deletion could regulate inflammatory stress and decrease inflammatory injury in hippocampal neurons, and consequently, ameliorate brain disorder [58]. Other studies suggest that TRPM2 may be involved in epilepsy through PARP1/BNIP3/AIF/Endo G signaling pathway in cortical neurons [59–61]. The production of inflammatory mediators (IL-1β, IL-6, TNF-α) and the expression of apoptosis related proteins (BNIP3, AIF and Endo G) in TRPM2 deficient epileptic mice are decreased [59]. In the current researches on TRPM2 and epilepsy, juvenile myoclonic epilepsy (JME) is a common idiopathic generalized epilepsy syndrome. Studies have shown that EFHC1 gene is closely related to JME [38, 40]. In situ hybridization analysis shows that TRPM2 and EFHC1 are co-expressed in hippocampal neurons and ventricular cells. The co-expression of TRPM2 and EFHC1 significantly enhances the production of hydrogen peroxide (H₂O₂) in HEK293 cells, and mediates the Ca²⁺ current by recombinant TRPM2 [38, 39]. In addition, EFHC1 enhances the susceptibility of TRPM2-mediated HEK293 cells to H₂O₂-induced cell death, while JME mutation reverses the susceptibility [39]. These results suggest that TRPM2 affects JME phenotype by mediating JME mutations in EFHC1 to disrupt biological processes including cell death [38–40]. In our study, we have found that TRPM2 knockout mice were more susceptible to epilepsy than WT. Furthermore, the neuronal excitability in hippocampal CA1 region of TRPM2 knockout mice is significantly increased. There are no significant differences in the input resistance and afterhyperpolarization of CA1 neurons in TRPM2 knockout mice and WT mice. What’s more, the firing adaptation rate of hippocampal CA1 pyramidal neurons of TRPM2 knockout mice was lower than that of WT. Our results showed that TRPM2-KO mice were more susceptible to epilepsy in the PTZ chronic kindling epilepsy model.

Given the critical role of potassium (K⁺) currents in regulating intrinsic electrical properties of neurons and their responsiveness to synaptic inputs, genetic research in the past two decades has identified several K⁺ channel genes as responsible for epilepsy. Neuronal Kv7 channels underlie a voltage-gated non-inactivating potassium current known as the M-current. Due to its particular characteristics, Kv7 channels show pronounced control over the excitability of neurons. Kv7 channel can regulate the intrinsic characteristics of CA1 hippocampal neurons by controlling resting membrane potential (RMP) and action potential threshold [62]. The KCNQ2/3 mutation causes M current to be down-regulated and the firing rate of neurons to be accelerated, leading to epilepsy. Present studies have shown that RMP can become smaller, and the number of action potentials can generate increased after Kv7 channel current is inhibited [62, 63]. Combined with the previous results, we found that TRPM2-KO neurons have a lower firing adaptation rate. Therefore, we considered that the increased excitability of hippocampal CA1 pyramid neurons in TRPM2-KO might be related to the Kv7 channel. To verify this hypothesis, we recorded Kv7 current directly and found that WT neurons had a significantly reduced Kv7 current with XE991, while TRPM2-KO not, which meant TRPM2-KO exactly had a deficit in Kv7 function. Besides, we found that activation of Kv7 channel reduces the firing frequency of action potential in the hippocampal pyramidal neurons of TRPM2 knockout mice. However, inhibiting Kv7 channel increased the firing frequency of action potential in hippocampal pyramidal neurons of WT mice. In animal experiments, we have confirmed that activation of Kv7 channel can effectively reduce epileptic seizures in TRPM2 knockout mice. These results suggest that hippocampal CA1 pyramidal neurons in TRPM2 knockout mice may increase neuronal excitability by inhibiting the Kv7 channel, thus affecting the susceptibility to epilepsy in mice.

To verify TRPM2-KO neurons may be inhibited through the Kv7 pathway to increase neuronal excitability, we adopted the PTZ acute epilepsy models (a single threshold dose: 50 mg/kg), considering uncontrollable factors may arise during the long-term drug administration. For example, it would be difficult to tell whether the corresponding blockers or agonist caused the differences. What’s more, the effects of the corresponding blocker or agonist on the epileptic seizures in mice can be observed simply and intuitionally by single administration of PTZ acute epilepsy model. However, this approach has obvious limitations. Due to the limitations of experimental conditions, time and other objective factors, accurate local intracranial administration cannot be achieved. In the acute model, intraperitoneal injection is still adopted, and the local effects of drugs on the brain cannot be determined by systemic administration. In addition, our results showed that TRPM2-KO mice were more susceptible to epilepsy in the PTZ chronic kindling epilepsy model. However, in the PTZ acute epilepsy model, the mortality of TRPM2-KO mice was significantly lower than that of WT group. Our previous reports showed that TRPM2-KO can effectively alleviate the nerve damage and neuroinflammation caused by epilepsy in pilocarpine epilepsy model [28]. Based on the findings, we proposed that TRPM2-KO mice may have higher neuronal excitability and may be more susceptible to epilepsy.

Firing adaptation refers to the phenomenon that the firing frequency of neurons decreases under the action potentials of continuous external stimulus. This is determined by the interaction of various ionic currents. Under current clamp, the neurons showed a repetitive firing pattern, and the firing frequency increased with the increase of the stimulus current. However, at the same stimulus current, the firing frequency will be slower during the repeated firing process (Fig. 3E). Mechanisms leading to this firing adaptation include M current, calcium ion mediated potassium ion conductance, and slow recovery from deactivation of fast sodium channels [64]. The latter two are closely related to afterhyperpolarization (AHP), and our study showed that there was no significant difference in mAHP between TRPM2-KO group and WT group at 290 pA (Fig. 3C, D). Therefore, we hypothesized that this difference in firing adaptation might be related to M current. This modulates the excitability of many peripheral and central neurons. Recent evidence suggests that the Kv7.2 and Kv7.3 subunits target the initial axonal segments of hippocampal neurons by binding to anchorin G. Furthermore, spontaneous mutations in these subunits impair the targeting of axons, leading to human neonatal epilepsy. Previous studies have reported that Kv7 channel is involved in several aspects of neuronal excitability, including resting membrane potential, peak frequency adaptability, and burst suppression [65–67]. Shah et al. reported that the function of axonal Kv7 channel in hippocampal CA1 pyramidal neurons is to inhibit the intrinsic spontaneous firing of neurons, which can be achieved by regulating the action potential threshold and RMP [62]. Our study also confirmed that Kv7 agonist can effectively reduce excitability of TRPM2-KO neurons and alleviate epileptic seizures in TRPM2-KO mice. Then we separated the Kv7 current with the application of XE991 and proved that TRPM2-KO exactly had a deficit in Kv7 function.

In conclusion, this study explores the mechanism why TRPM2 knockout mice are more susceptible to epilepsy and finds that TRPM2 knockout mice may have a deficit in Kv7 function. We believe that TRPM2 may be a new therapeutic target for epilepsy. According to our results, to reduce the excitability of neurons from the level of gene or ion channel may be crucial to reduce abnormal discharge, the incidence of epilepsy and the probability of genetics, and to provide a new idea for the treatment of epilepsy.

Through the study of this subject, we found that in the model of PTZ kindling epilepsy, TRPM2 gene knockout mice were more susceptible to epilepsy than WT mice. Besides, hippocampal CA1 pyramidal neurons in TRPM2 knockout mice may increase neuronal excitability by inhibiting the Kv7 channel, therefore affecting the susceptibility to epilepsy in mice.

Authors’ Contributions

Peifang Jiang and Tao Zhu designed the experiments. Yingchao Ying, Lifen Gong, and Chen Chen performed the experiments. Yingchao Ying and Peifang Jiang analyzed and wrote the manuscript. Other authors helped perform the experiments. All authors contributed to the article and approved the submitted version.

AP Action potential

CNS Central nervous system

Kv7 KCNQ-encoded voltage-dependent K⁺ channels

mAHP Medium after hyperpolarization

M current Kv7 current

PTZ Pentylenetetrazole

RMP Resting membrane potential

Rin Input resistance

ROS Reactive oxygen species

sEPSC Spontaneous excitatory postsynaptic current

TRPM2 Transient receptor potential melastain2

Authors’ Contributions

Funding

This work was supported by Grants from the National Natural Science Foundation of China (No. 82171438, 81671287, 81372116, 81201511), Health Innovative Talents in Zhejiang Province, and Zhejiang Province Public Welfare Technology Application Research Project (No. LY22H090005, LY15H090006).

Data Availability

The datasets used during the present study are available from the corresponding author upon reasonable request.

Compliance with Ethical Standards

The animal study was reviewed and approved by the Animal Experiment Ethics Committee of Zhejiang University.

Acknowledgments

The TRPM2 KO mice were kindly provided by Prof. Fang Xiangming from Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Japan. We are grateful to Prof. Zhou Yudong who helped us to successfully complete this research work.

Conflict of Interest The authors declare that they have no conflict of interest.

Consent to Participate Not applicable.

Consent for Publication All authors consent to the publication of this manuscript.

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TRPM2 regulates the excitability of hippocampal neurons through Kv7 channel in epilepsy

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Abstract

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Introduction

Materials And Methods

Animals

Pentylenetetrazol treatment

Brain slice preparation

Electrophysiology recordings of brain slices

Statistical Analysis

Results

TRPM2 knockout is more susceptible to seizures in PTZ chronic kindling epilepsy model

The adaption rate of hippocampal CA1 pyramidal neurons in TRPM2-KO was lower

The increased excitability of TRPM2-KO neurons is related to the Kv7 channel

Discussion

Conclusion

Authors’ Contributions

Abbreviations

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References

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