Absence epilepsy is a multifactorial epileptic syndrome that can be acquired or inherited. Absence seizures are identified as diffuse, generalized, non-convulsive seizures associated with cessation of activity and a transient change in consciousness with bilateral, synchronous, and regular 3-Hz spike-wave discharge (SWD) activity on electroencephalogram (EEG) (Caplan et al., 2008). Among the frequently used experimental models that meet the criteria for absence-type, non-convulsive seizures is the Wistar Albino Glaxo/Rijswick (WAG/Rij) rat with spontaneous bilateral synchronous SWD activities observed in cortical EEG recordings with a frequency ranging from 7 to 11 Hz, an amplitude spanning from 100 to 450 μV, and an average duration of 5 seconds (with a range of 1–30 seconds) (Coenen and Van Luijtelaar, 2003; Russo et al., 2016). These animal models carry behavioral and characteristic features of human absence epilepsy (Sitnikova and van Luijtelaar, 2007).

Thalamocortical communication regulates oscillations, the source of SWD activity. The thalamus and cortex play roles in the abnormal rhythmic patterns observed in generalized epilepsies, such as absence and myoclonic seizures. These patterns involve aberrant synchronization and oscillatory behavior between thalamic and cortical regions, facilitating the generation and spread of epileptic activity (Kostopoulos, 2001; Lindquist et al., 2023). In the pathophysiology of absence epilepsy, the ventrobasal nucleus (VB) of the thalamus and thalamic reticular nucleus (TRN) is crucial for SWD formation (Avoli and Gloor, 1982; Danober et al., 1998; Blumenfeld, 2005). TRN regulates thalamocortical oscillations and facilitates information flow between the thalamus and cortex (Avanzini et al., 1993). This study examined molecular changes in the thalamus and cortex tissues separately to highlight their essential roles in the occurrence and progression of SWD.

Increased GABAergic (Ɣ-aminobutyric acid-ergic) activity in TRN leads to the activation of low-voltage activated T-Type Ca2+ channels through prolonged hyperpolarization (Avanzini et al., 1993; Manning et al., 2003). Ca2+ ions entering through these channels trigger bursts of Na + -dependent action potentials, causing membrane depolarization and low-threshold spikes (Huguenard and Prince, 1992; Perez-Reyes, 2003). T-type Ca2+ channels, including Cav3.1 (CACNA1G), Cav3.2 (CACNA1H), and Cav3.3 (CACNA1I), play a crucial role in generating burst-type abnormal oscillatory activity in the thalamocortical loop. Cav3.2 driven bursts are significant for absence propagation in TRN. Mutations in CACNA1H may increase seizure susceptibility by affecting Ca2+ -regulated transcription factors involved in neuron development and gene expression (Eckle et al., 2014).

The endoplasmic reticulum (ER), including folding proteins, is vital for cellular function. Accumulation of misfolded proteins triggers the unfolded protein response (UPR), reducing translation and prompting refolding, eventually leading to endoplasmic-reticulum-associated protein degradation (ERAD) (Dobson et al., 1998). UPR is regulated by three sensors: protein kinase R (PKR)–like endoplasmic reticulum kinase (PERK) (Harding et al., 1999), inositol requiring enzyme 1α/β (IRE1) (Cox et al., 1993; Mori et al., 1993), and activating transcription factor 6α/β (ATF6). UPR-targeted gene upregulation enhances the translation of regulatory protein exclusively within the UPR signaling pathway (Guan et al., 2014; Adams et al., 2019).

78-kDa glucose-regulated protein (GRP78), a marker of ER stress, acts as a chaperone beyond UPR, maintaining ER homeostasis by reducing misfolded proteins (Park et al., 2021). Under ER and calcium stress, the GRP78 level rises (Resendez et al., 1985; Suzuki et al., 1991). Irrepressible ER stress triggers apoptotic signals like C/EBP homologous protein (CHOP), aka DNA damage-inducible gene 153 (GADD153) (Oyadomari and Mori, 2004). GADD153 responds to various stresses, inducing apoptosis (Marciniak et al., 2004; Li et al., 2009).

ER stress can be targeted pharmacologically in two ways: the drugs can address misfolded protein accumulation or modulate chaperons (Marciniak et al., 2022). UPR plays a role in cancer, diabetes, and neurodegenerative diseases. Small molecules (chemical chaperones) targeting UPR pathways have been developed to treat ER-associated diseases, maintain protein homeostasis, and prevent neurodegeneration. While many small molecules are in pre-clinical studies, bortezomib, which enhances ER stress to kill cancer cells, is in human trials (Manasanch and Orlowski, 2017).

Thapsigargin (Tg), a sesquiterpene lactone derived from Thapsia garganica, is a tumor initiator that disrupts Ca2+ balance via sarco/endoplasmic reticulum calcium ATPase (SERCA) in the ER membrane, which is crucial for cellular stress (Sagara et al., 1992). The purpose of choosing Tg as an ER stress agent is to induce intracellular calcium imbalance, a global stressor for cells, which impacts T-type calcium channels such as Cav3.2, associated with SWDs, by selectively binding SERCA (Hiroi et al., 2005; Cain et al., 2018).

Although current anti-epileptic treatments provide complete seizure control in most patients with epilepsy, approximately 30% of patients develop refractory epilepsy that does not respond to these treatments (Billakota et al., 2020). The role of ER stress in epileptogenesis is gaining attention, and pre-clinical studies are exploring its effects and potential neuroprotective strategies, including anti-epileptic drugs (Fu et al., 2019; Liu et al., 2019). A recent study has demonstrated that sodium valproate (VPA) reduces GRP78, CHOP protein expressions, and neuronal apoptosis in the pentylenetetrazol (PTZ)-induced epilepsy model (Fu et al., 2019). Another study mentioned that acute Tg-induced ER stress suppresses neuronal activity via a protein translation-dependent mechanism that down-regulates p53 (a tumor suppressor gene) and indicated that inhibition of ER stress may be a neuroprotective strategy for seizures (Liu et al., 2019). Understanding the effect of ER stress on epileptogenesis at the molecular level may enable the discovery of new and more effective options for the treatment of epilepsy. Therefore, this study aimed to investigate the role of ER stress induced by Tg in regulating SWD activity in the WAG/Rij model and to evaluate the possible contribution of molecular pathways of ER stress to epileptogenesis, CACNA1H, and immune responses (NF-κB and TNF-α) in thalamus and cortex tissues.