The term epileptogenesis is defined as a sequence of events that convert a normal circuit into a hyperexcitable circuit (Pitkänen et al., 2015). Epileptogenesis refers to the development of tissue capable of generating spontaneous seizures, which result in an epileptic condition or in the progression of established epilepsy (Pitkänen and Engel, 2014), whereby it is a continuous and multifactorial process in which the eCBs participate in the modulation of neuronal activity, neuronal migration, axonal growth and guidance, synaptic plasticity, and the neuroinflammatory response (Schonhofen et al., 2018; Cheung et al., 2019). It has been established that damage to eCB signaling in the early stages of neurodevelopment can favor the process of epileptogenesis and the establishment of epilepsy in later stages (Figure 2).

During the latent period, the participation of eCBs has been described in different cascades of events defined as acute, subacute, and chronic (Kadam et al., 2010). The induction of early genes (IEGs), including Fos, Jun, Egr4, Egr1, Homer 1, Nurr77, and Arc, occurs during acute changes as a result of intense and repeated synaptic activity during seizure activity, which promotes increased intracellular Ca²⁺ flux by NMDARs (Herdegen and Leah, 1998). Upregulation of IEGs can pathologically modulate synaptic function, lowering the threshold to neuronal hyperexcitability (Rakhade and Jensen, 2009). The levels of intracellular Ca²⁺ immediately alters synaptic efficiency mechanisms, such as increasing the postsynaptic density and dendritic spines, grouping NMDARs and AMPARs, and increasing glutamate synthesis (Haglid et al., 1994; Sanchez et al., 2001), where the participation of eCBs plays an important pathophysiological role in modifying excitatory and inhibitory synaptic neurotransmission in the brain (Rosenberg et al., 2017). During postnatal neurodevelopment, CB1R located in presynaptic cells, mainly in the granule cells of the hippocampus, regulates excitatory activity; additionally, endocannabinoids can also act through interactions with other types of receptors, mainly on G protein-coupled receptors, such as GPRSS and the Transient receptor potential (TRP) cation channel subfamily V member 1" or “vanilloid receptor 1” (TRPV1), which participate in neuronal hyperexcitability, favoring intracellular Ca²⁺ flux (Bhaskaran and Smith, 2010; Schonhofen et al., 2018; Cheung et al., 2019).

As we have indicated, the synthesis of endocannabinoids is mediated by an increase in intracellular Ca²⁺ generated by neuronal hyperexcitability and the demand for membrane phospholipid diacylglycerol lipase (DAGL) and for glutamate and acetylcholine (Wallace et al., 2003; Rosenberg et al., 2017). The eCBs passively diffuse through presynaptic cells in a retrograde manner to bind orthosterically to and activate CBR1 acylphosphatidyl ethanolamine-hydrolyzing phospholipase D (NAPE-PLD) to form 2-AG and AEA, in the same way, endocannabinoids are also synthesized from the activation of metabotropic receptors and thus inhibit the release of glutamate or GABA, a process defined as depolarization-induced suppression of inhibition (DSI) or excitation (DSE), respectively (Armstrong et al., 2009). In models of febrile seizures at PN day 10, alterations to these DSI and DSE mechanisms have been reported, which are estimated to interfere with the maturation of the GABAergic system and persist in later stages through the generation of hyperexcitable circuits (Bernard et al., 2005).

Similarly, it has been reported that eCB signaling has an important role in epileptogenesis, because, after the dissociation from CB1R, eCB 2-AG and AEA are catabolized by the enzymes monoacylglycerol lipase (MAGL) and alpha-beta hydrolase domain containing 6 (CABHD6) or through fatty acid amide hydrolase (FAAH), respectively, and activation of TRPV1 by AEA can trigger increased glutamate release after intracellular Ca²⁺ concentrations are increased (Wallace et al., 2003; Rosenberg et al., 2017). This process activates calcineurin (PPP3C or serine-threonine protein phosphatase 2B), which, during intense seizure activity, activates calcium-calmodulin-phosphatase to promote the dephosphorylation and subsequent endocytosis of GABA_A (Blair et al., 2004). These phenomena reduce the inhibitory potential and lead to the gradual loss of GABAergic inhibitory networks (Kurz et al., 2001; Blair et al., 2004; Rakhade and Jensen, 2009; Semple et al., 2020). In the immature brain, calcineurin activation increases the phosphorylation of Kv2.1 ion channels (also known as KCNB1) and promotes their expression in the postsynaptic membrane, allowing prolonged neuronal depolarization (Kurz et al., 2001; Rakhade and Jensen, 2009).

Similarly, protein kinase C, which is dependent on protein kinase type II and calcium-calmodulin-phosphatase, increases within minutes after the induction of SE in immature rats, which allows an increase in the phosphorylation of serine 831 of GluR1 and serine 880 of GluR2. This process promotes the endocytosis of GluR2 and increases the permeability to Ca²⁺ in AMPARs, which generates an increase in seizure susceptibility in later or adult stages (Rice and De Lorenzo, 1998; Sanchez et al., 2001; Rakhade and Jensen, 2009). Additionally, it has been reported that astrocyte activation, immediate to neuronal damage, increases extracellular K⁺ concentrations, which in turn facilitates continuous neuronal hyperexcitability, a triggering factor for epileptogenesis (Jabs et al., 1997; Jensen, 2009).

Subacute changes in the latent period are established from hours to days after neuronal damage (Figure 2), in which a rapid increase occurs in NGF and BDNF, which together with Trk alter the modulation of the maturation of the KCC2 cotransporter, promoting GABA-mediated inhibitory activity mainly in DG granular cells, which in turn alters the modulating function of eCBs on the GABAergic system to favor long-term neuronal hyperexcitability (Chen et al., 2003, 2007). However, the cellular mechanisms by which CB1R and CB2R act on short- and long-term changes in the seizure threshold are still unknown, particularly in mesiotemporal structures consisting of mainly the hippocampus and the cerebral cortex in the immature brain (Chen et al., 2007).

The neuroinflammatory response activated by microglia and astrocytes, the main response responsible for the synthesis and release of pro-inflammatory cytokines such as IL1-β, activates the IL-1/TLR signaling pathway, where an increase in pro-inflammatory proteins occur. These proteins include Cox-2, an enzyme that catalyzes the production of PGs from AA and increases seizure susceptibility (Chen et al., 2001, 2007; Jiang et al., 2004; Feng et al., 2016), and TNF-α, which generates an increase in the expression of AMPAR and a decrease in GluR2 through the expression of TNF-R1 on the neuronal membrane surface, which in turn causes increased permeability of immature neurons to Ca²⁺ (Beattie et al., 2002; Stellwagen et al., 2005). The activity of TNF-α has been shown to result in the endocytosis of GABA_A receptors, mainly their β 2/3 subunits, in the pyramidal neurons of CA1 and interneurons of the DG, promoting neuronal excitation (Wang et al., 2000; Balosso et al., 2005; Stellwagen et al., 2005; Shimada et al., 2014) and leading to neurodegeneration and neuronal death in the CA1 and CA3 regions of the immature hippocampus (Rizzi et al., 2003; Kawaguchi et al., 2005).

In the same way, the rapid increase in Cox-2 mRNA expression after SE induced by KA itself induces neurodegeneration and neuronal death in the immature hippocampus, mainly the CA1 and CA3 subfields, at 12 and 24 h post SE (De Simoni et al., 2000; Rizzi et al., 2003; Kawaguchi et al., 2005). This finding supports that the neuroinflammatory response precedes neuronal damage (Ravizza et al., 2005; Joseph and Levine, 2006), and this neuronal damage is associated with an increase in Cox-2 and the subsequent production of PGs and ROS, activation of the oxidative stress mechanism, and mitochondrial dysfunction (Gobbo and O’Mara, 2004); thus, it has been reported that the eCBs participate as a substrate of the Cox-2 enzyme for the synthesis of PGs (Nomura et al., 2011; Ruhaak et al., 2011). Similarly, experimental models of SE induced by KA and Li-Pilo in PN 12 day rats have reported a rapid increase in ROS and mitochondrial markers of oxidative damage, including NT-3, 4-HNE and carbonylated proteins, as well as the formation of free radicals 24 h post-SE, which are responsible for neurodegeneration in the subfields of CA1, CA3 and DG of the hippocampus, cerebral cortex and thalamus (Patel and Li, 2003; Folbergrová et al., 2010).

The contribution of mitochondrial dysfunction on complex I of the respiratory chain NADH (Cock, 2002; Kudin et al., 2002) favor the development of neurodegenerative disorders, such as epilepsy, due to the excessive release of glutamate, which causes an alteration in the reuptake mechanisms of this neurotransmitter and an increase in intracellular Ca²⁺ due to the saturation of the regulatory mechanisms mediated by the Na⁺/Ca²⁺ exchanger and the Ca²⁺ buffer proteins (Tretter et al., 2004).This causes the mitochondria to capture the excess Ca²⁺ in the mitochondrial matrix, which induces the depolarization of the membrane by the partial inhibition of the chemiosmotic potential and by the accumulation of positive charges in the mitochondrial matrix (Tretter et al., 2004). This sustained overload produces an irreversible depolarization through the activation of the mitochondrial transition pore, a pathway through which Ca²⁺ returns to the cytosol, causing the collapse of the mitochondrial chemiosmotic potential and a reduction in the synthesis of ATP (Murchison and Griffith, 2000). The decrease in ATP generates metabolic dysfunction, ROS production, activation of proteases, phospholipases, iNOS, and endonucleases, and inhibition of protein synthesis. Furthermore, NO can act as a retrograde messenger, enhancing the excitotoxic effect of glutamate and increasing its release from presynaptic terminals (Lorigados et al., 2013). Likewise, it has been reported that complex I dysfunction may also be the result of an increase in carbonylated proteins during the subacute phase post-SE induced by Li-Pilo and that this alteration persists up to 5 weeks, a period in which spontaneous convulsive activity is observed (Folbergrová et al., 2010).

Likewise, Cox-2 and TNF-α act directly on receptors and ion channels and indirectly by modulating extracellular glutamate reuptake systems via the reduction of selective transporters to glutamate GLT-1, mediated by astrocytes, mainly in CA1 and DG of the hippocampus. Additionally, the activation of iNOS, which promotes the synthesis of NO, increases the release of glutamate and substance P, which preserves the activation of cytokines together with the increase in the affinity of NMDARs and AMPARs, which supports the finding that the activation of the neuroinflammatory response by microglia and astrocytes increases the vulnerability of the immature hippocampus to neuronal hyperexcitability and that these changes depend on the age of development (De Simoni et al., 2000; Rizzi et al., 2003; Viviani et al., 2003; Ravizza et al., 2005; Stellwagen et al., 2005; Bessis et al., 2007; Chen et al., 2013). The participation of CB2R, which is overexpressed by microglia in the neuroinflammatory response in response to neuronal damage and the infiltration of cells of the immune system into the brain parenchyma (Sagredo et al., 2009; Bouchard et al., 2012), induces chronic CB2R activation, which increases excitatory neurotransmission (Li and Kim, 2015) and decreases inhibitory neurotransmission (Morgan et al., 2009). The CB2R, which are mostly expressed in microglia, have been reported to participate in the neuroinflammatory response in experimental models in rats, where they increase 2-AG and CB1R in the hippocampus after SE (Wallace et al., 2003); however, the increase in 2-AG in the seizure model in mice shows a decrease in seizure activity (Sugaya et al., 2016).

Chronic changes (Figure 2) can be observed over 2–12 weeks in rodents and from months to years in humans. It has been reported that during the first 2 weeks of life, the resistance to excitotoxic damage in the immature brain is relative since the amount of Ca²⁺ that enters a pyramidal neuron is directly related to the age of PN development, where in the first 3 days of life, glutamate minimally increases intracellular Ca²⁺. Conversely, between PN days 10 and 25, intracellular Ca²⁺ increases markedly due to recurrent seizure activity, leading to neuronal death in the CA1 subfield of the hippocampus, the area most vulnerable to excitotoxic damage, due to an increase in NMDARs and AMPARs (Kubová et al., 2001, 2012; Kubová and Mareš, 2013). Additionally, an alteration in the immature glutamatergic system that results in the overexpression of GluR2 and its activity on the intracellular Ca²⁺ flux through AMPARs and KARs, a condition that pathologically persists during the adolescent stage mainly in the prefrontal cortex, favors the formation of hyperexcitable circuits. Similarly, prolonged positive regulation has been reported in the expression of the CB1R and in the suppression of the inhibition induced by depolarization, which can cause alterations in neuronal hyperexcitability throughout life (Chen et al., 2003; Bernard et al., 2005).