There are extensive high-frequency oscillations (HFOs) in neural networks. The term HFOs refers to electroencephalogram (EEG) activity with a frequency of 40–500 Hz, including γ oscillation (40–80 Hz), ripple oscillation (80–200 Hz), and fast ripple oscillation (250–500 Hz) (Höller et al., 2019). HFOs can be divided into physiological and pathological, which differ in their frequency, location and mechanisms (Le Van Quyen et al., 2006). Physiological HFOs gradually mature with brain development. Ripples (>140 Hz) are observed in the hippocampus of rats during their second week after birth. A vitro hippocampal model has confirmed that physiological HFOs can be controlled by a feedback circuit between GABAergic interneurons and pyramidal cells (Hájos and Paulsen, 2009). In physiological states, HFOs are associated with sensory information processing and hippocampal memory function (Höller et al., 2019); whereas, in pathological states, HFOs are closely related to epilepsy (Frauscher et al., 2017; Velmurugan et al., 2019; Tingley and Buzsáki, 2020).

Our previous study showed that the energy of HFOs in hippocampal regions CA1 and CA3 of the lithium-pilocarpine (PILO) status epilepticus (SE) model was significantly higher than that of physiological HFOs (Song et al., 2016). The spectral power of fast ripples (FRs) during seizures can be used as a quantitative indicator providing an early warning of seizures (Song et al., 2016).

The generation mechanism of HFOs is not completely clear; HFOs may be caused by a variety of mechanisms rather than a single mechanism of neural cells and networks. At present, it is believed that pathological HFOs, whether ripples or FRs, mainly reflect the action potential of principal cells. It may be related to the gap junction (GJ) network (Jiruska et al., 2017). Gap junctions are intercellular channels composed of special transmembrane proteins. These transmembrane protein families are called connexins (CXs) (Beyer and Berthoud, 2018). At present, more than 20 kinds of CXs have been found, and CX26 is expressed in a variety of nerve cells, especially neurons (Su et al., 2017); CX32 is widespread on oligodendrocytes (Men et al., 2019). CX36 is a connexin preferentially expressed by neurons, and it plays an important role in the transmission of electrical signals (Li et al., 2019). CX43 has the highest expression in astrocytes and it may be involved in the regulation of nerve injury, as well as epileptogenesis (Deshpande et al., 2017). The increase in electrical coupling between GJs transforms physiological HFOs into pathological HFOs (Stacey et al., 2011), which induce synaptic plasticity (Jefferys et al., 2012). Our previous study confirmed that in an acute epilepsy rat model, the spectral power of FRs and the degree of seizures can be downregulated by inhibiting the expression of different CXs (Ran et al., 2018).

It is not known whether connexins participate in the occurrence of pathological HFOs, thereby establishing abnormal electrosynaptic transmission and increasing susceptibility to seizure in the chronic phase of epilepsy. Therefore, we established an chronic epilepsy rat model to observe dynamic changes in the energy of HFOs and the expression of CXs in the hippocampus, to explore the role of GJ receptor blockers in the chronic phase of epilepsy, and to clarify whether GJ plays a key role in the occurrence and development of epilepsy, in order to provide a theoretical basis for the selection of new targets for antiepileptic therapy.

The overall protocol and time-course of the evaluation of various outcomes are shown in Figure 1A.

Pilocarpine is a post-ganglionic cholinergic drug, which can directly stimulate M-cholinergic receptors to produce a cholinergic effect, thereby inducing seizures by activating central cholinergic receptors. The adult rat lithium-pilocarpine epilepsy model exhibits three periods: an acute phase–6–24 h after SE; a latent phase–no organized activity and EEG for at least 1 day; and a chronic phase–the appearance of SRSs. SRSs are important clinical manifestations of epilepsy. In the rat model of epilepsy, which is created using lithium-pilocarpine, SRSs appear after SE. The damage and manifestations of this model are similar to human temporal lobe epilepsy, and this model is one of the most commonly used experimental epilepsy models (Song et al., 2016). In this study, SRSs occurred within 16 days after SE, indicating entry into the chronic phase of epilepsy in the experimental rats.

Enhanced intercellular GJ communication may be involved in epileptic production (Mylvaganam et al., 2014). CBX is a glycyrrhizonic acid derivative that reduces plasma membrane mobility and inhibits gap junction conductance through multiple pathways, including protein kinase, G protein, transport ATP enzyme, and CX phosphorylation (Walrave et al., 2020). It is a broad-spectrum GJ blocker that acts on a variety of CXs. Whether in cell culture in vitro, acute brain slices, or in vivo animal experiments, CBX has been proven to block GJ quickly and reversibly, thus, further reducing the number of spontaneous/induced seizures, and reducing the duration, frequency, and amplitude of epileptic discharges (Vincze et al., 2019; Walrave et al., 2020). CBX can reduce the frequency of spontaneous HFOs activity (Naggar et al., 2020). This study confirmed that CBX can reduce the number of SRSs and the average spectral power of FRs in rats, with an antiepileptic effect.

High-frequency oscillations in the human brain are affected by sleep. Studies show that the rate of HFOs is highest during non-rapid eye movement (NREM) sleep and lowest during rapid eye movement (REM) sleep and the waking stage; the area of HFOs during NREM sleep is larger (von Ellenrieder et al., 2017). Our previous study showed that physiological and pathological HFOs have similar sleep balance characteristics (Yan et al., 2021). At present, the correlation between HFOs and sleep in the animal brain is not clear. Therefore, in our study, we selected 10-min samples of EEG signals during the interictal period (waking stage) to explore the effect of CBX on the energy changes of ripples and FRs in rats with chronic epilepsy.

High-frequency oscillations are generated by multiple mechanisms such as synchronized inhibitory post-synaptic potentials with sparse pyramidal cell firing or principal cell action potentials (Ylinen et al., 1995; Bragin et al., 2011). Synchronization of fast firing within the population of interconnected neurons leads to the formation of an episode of high-frequency population spikes, which is extracellularly recorded as an HFO event. It requires synchronization on a millisecond time scale, which is achieved via fast synaptic transmission or non-synaptic mechanisms like gap-junction coupling or ephaptic interactions–a synchronizing mechanism that depends on specific geometric organization and tight cellular arrangement (Jiruska et al., 2017). Both pyramidal neurons and interneurons are involved in HFO generation, but pyramidal cells fire preferentially at the highest amplitude of the ripples, and interneurons begin to discharge earlier than the pyramidal cells do (Cepeda et al., 2020). The inhibitory effect of the interneurons is maintained in ripples (Jefferys et al., 2012), whereas FRs reflect hypersynchronous population spikes of excitatory pyramidal cells (Kanazawa et al., 2015). The formation mechanism of HFOs with different frequencies may be different. The ripples may be formed by the synchronization of the inhibitory post-synaptic potential generated by excitatory neurons mediated by GABAA receptors, while the FRs may be derived from the field potential formed by transient, highly synchronized and sudden discharges of excitatory neurons with pathological connections (Jacobs et al., 2014; Frauscher et al., 2017). Our previous studies confirmed that the frequency of HFOs is not necessary to distinguish physiological and pathological states, that ripples and FRs can be present in both the normal and the epileptic hippocampus, and that the main difference between the two states is the different energy of HFOs (Ran et al., 2018). This study found that during the chronic phase of epilepsy, the average and/or peak spectral power of ripples did not significantly change before and after SE in the PILO and CBX + PILO groups. However, the average and peak spectral power of FRs in the PILO group were significantly higher at 1 and 7 days after SE than those 1 day before SE, and that the average spectral power of FRs in the PILO group were significantly higher than those in CBX + PILO group at 1, 7, and 14 days after SE. This suggests that in the chronic phase of epilepsy, FRs respond more to the real situation of SRSs than ripples do. In a study of intracranial EEG in epileptic patients, González Otárula et al. (2019) found the distribution of ripples was widespread, while FRs were restricted to the epilepsy initiation region. The association of FRs with seizure onset zone may be stronger than that of ripples (Frauscher et al., 2018). Animal studies suggest that ripples are related to physiological functions, such as the formation of memory and cognition, while FRs are related to the seizure onset zone (Nevalainen et al., 2020). FRs are able to influence the specific synaptic driving function of CA1 pyramidal cells, make neuronal firing randomized, and ultimately lead to the loss of control of selective discharges in the hippocampus (Valero et al., 2017). It has been suggested that an energy analysis of FRs may be a more sensitive and specific biomarker of epilepsy, compared to ripples.

Gap junction is considered to be an important component of the neuronal network, with synchronized neuronal activity and field potential oscillations (Posluszny, 2014). The EEG signals during the seizure and interictal seizure phases are correlated with the degree of electrical coupling to the GJ (Roopun et al., 2010). The upregulation of CX43 by the transient receptor potential vanoxalate-4 (TRPV4) may be involved in the pathophysiological process of epilepsy (Men et al., 2019), and the specific CX43 mimic peptide and TAT-Gap19 can reduce spontaneous seizures by inhibiting the function of GJ channels/hemi-channels of CX43 between astrocytes (Delvaeye et al., 2018; Walrave et al., 2018). We investigated the effects of seizures and CBX intervention on the expression of different CXs in the hippocampus in chronic epilepsy and found that seizures induced an increase in CX43 expression at 1 and 7 days after SE, but had no significant effect on CX26, CX36, or CX32. CBX pretreatment did not affect the expression of CXs in the hippocampus of normal rats, but it inhibited CX43 expression in epileptic rats. Further analyses revealed that the expression of CX43 was more strongly correlated with the spectral power change of FRs than the other three CXs, suggesting that the energy of FRs may be regulated by interfering with the expression of CX43, so as to affect seizures. Therefore, in addition to traditional antiepileptic drugs, drugs should be developed that target blocking the electrical conduction of GJ, thereby reducing pathological HFOs formation, which can provide a new strategy for treating epilepsy. However, the causal relationship between the energy of FRs and the expression of CX43 is still unclear. What specific signaling pathways of connexin regulate HFOs, or how neuroelectrical activity affect connexin expression, remain to be explored in the future.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Ethics Committee of Chongqing Medical University.

YH conceived and designed the experiments and revised the manuscript critically for important intellectual content. BL made acquisition and interpretation of data and was involved in drafting the manuscript. BL, XR, and YY performed the experiments. XZ and HC performed data analysis. All authors wrote the manuscript and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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