Progressive Seizure Aggravation in the Repeated 6-Hz Corneal Stimulation Model Is Accompanied by Marked Increase in Hippocampal p-ERK1/2 Immunoreactivity in Neurons

Abstract

The 6-Hz corneal stimulation test is used to screen novel antiepileptic molecules to overcome the problem of drug refractoriness. Although recognized as a standard test, it has been evaluated only recently in the attempt to characterize the putative neuronal networks involved in seizures caused by corneal stimulation. In particular, by recording from the CA1 region we previously established that the hippocampus participates to propagation of seizure activity. However, these findings were not corroborated by using markers of neuronal activation such as FosB/ΔFosB antigens. In view of this discrepancy, we performed new experiments to characterize the changes in levels of phosphorylated extracellular signal-regulated kinases1/2 (p-ERK1/2), which are also used as markers of neuronal activation. To this aim, mice underwent corneal stimulation up to three different times, in three sessions separated by an interval of 3 days. To characterize a group in which seizures could be prevented by pharmacological treatment, we also considered pretreatment with the ghrelin receptor antagonist EP-80317 (330 μg/kg). Control mice were sham-treated. Video electrocorticographic (ECoG) recordings were obtained from mice belonging to each group of treatment. Animals were finally used to characterize the immunoreactivity for FosB/ΔFosB and p-ERK1/2 in the hippocampus. As previously shown, FosB/ΔFosB levels were highly increased throughout the hippocampus by the first induced seizure but, in spite of the progressively increased seizure severity, they were restored to control levels after the third stimulation. At variance, corneal stimulation caused a progressive increase in p-ERK1/2 immunoreactivity all over the hippocampus, especially in CA1, peaking in the third session. Predictably, EP-80317 administration reduced both duration and severity of seizures, prevented the increase in FosB/ΔFosB levels in the first session, and partially counteracted the increase in p-ERK1/2 levels in the third session. The vast majority of p-ERK1/2 immunopositive cells were co-labeled with FosB/ΔFosB antibodies, suggesting the existence of a relationship between the investigated markers in a subpopulation of neurons activated by seizures. These findings suggest that p-ERK1/2 are useful markers to define the aggravation of seizures and the response to anticonvulsant treatments. In particular, p-ERK1/2 expression clearly identified the involvement of hippocampal regions during seizure aggravation in the 6-Hz model.

6 Hz corneal kindling in mice triggers neurobehavioral comorbidities accompanied by relevant changes in c‐Fos immunoreactivity throughout the brain

“…Moreover, 4 repeated corneal stimulations (6 Hz, 32 mA) evoked seizures, which appeared first in the frontal cortex and then spread to the hippocampus, suggesting the establishment of an epileptogenic process. In this paradigm a significant increase in neuronal activity was observed in the lateral amygdala after the fourth session, and in the hippocampus after the first session, which vanished to control levels after repeated corneal stimulations . Consistent with the previous findings, we observed elevated c‐Fos immunoreactivity in the amygdala, hippocampus, and Cg.…”

Modeling and treatingGRIN2Adevelopmental and epileptic encephalopathy in mice

“…Specifically, the relative resistance of Grin2a S644G heterozygotes to 6 Hz partial seizures suggests altered limbic system function or connectivity (Barton et al, 2001;Metcalf et al, 2017). Ironically, vulnerability in the limbic system is also suggested by the lethal seizures whose features include a wild run followed rapidly by tonic-clonic episodes and tonic hind limb extension, resembling audiogenic seizures which are known to involve paroxysmal activity in the cortex, medial geniculate, hippocampus, and amygdala (Marescaux et al, 1987;Naritoku et al, 1992;Dutra Moraes et al, 2000;Faingold, 2012); the main structures of limbic circuitry (Giordano et al, 2015;Giordano et al, 2016) Functional characterization of mutant channel activity shows a very strong gain-of-function effect and slower kinetics, supported by assessment of synaptic activity in excitatory neurons of mutant mice. Evaluation of receptors with 0, 1, or 2 copies of the mutant allele revealed an intermediate effect of S644G when only one copy of the variant is present in the complex.…”

Effects of arachidonyl-2’-chloroethylamide (ACEA) on the protective action of various antiepileptic drugs in the 6-Hz corneal stimulation model in mice

“…In our study, there was no need to differentiate between convulsive and non-convulsive psychomotor signs in mice in the 6-Hz corneal stimulation model because, as documented earlier both, convulsive and non-convulsive components correspond to evident pathological changes in the brain electrical activity in experimental mice. More specifically, it has been observed during the EEG recording that both, minimal stereotypies and stunned posture in mice, defined as main non-convulsive signs in the 6-Hz corneal stimulation model, reflected flattering and post-ictal depression of the brain electrical activity in animals [ 37 , 38 ]. This was the reason to select a recovery to normal behavior within 20 seconds as a criterion that clearly defined a state without any abnormal electric activity in the brains of the mice in the EEG recording.…”

“…After application of ocular anesthetic (0.5% solution of tetracaine hydrochloride) to the mouse corneas, the animals underwent corneal stimulation and were placed separately in Plexiglas cages (25 × 15 × 10 cm) for the observation of the presence or absence of psychomotor seizures, as described previously [ 14 , 15 , 35 , 36 ]. Immediately following the 6-Hz corneal stimulation the animals exhibited a “stunned” posture associated with rearing and automatic movements (convulsive and non-convulsive components) that lasted from 60 to 120 s in untreated animals [ 29 , 30 , 37 , 38 ]. In this study, seizure activity associated with the 6-Hz corneal stimulation in mice was characterized by both, convulsive and non-convulsive components, including, immobility or stun, jaw and forelimb clonus, twitching of the vibrissae, and an elevated tail or Straub-tail, as mentioned elsewhere [ 29 , 30 , 37 , 38 ].…”

“…Immediately following the 6-Hz corneal stimulation the animals exhibited a “stunned” posture associated with rearing and automatic movements (convulsive and non-convulsive components) that lasted from 60 to 120 s in untreated animals [ 29 , 30 , 37 , 38 ]. In this study, seizure activity associated with the 6-Hz corneal stimulation in mice was characterized by both, convulsive and non-convulsive components, including, immobility or stun, jaw and forelimb clonus, twitching of the vibrissae, and an elevated tail or Straub-tail, as mentioned elsewhere [ 29 , 30 , 37 , 38 ]. After the seizure activity, the animals resumed their normal exploratory behavior.…”