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.

Keywords: 6-Hz corneal stimulation; EP-80317; FosB; electrocorticography; epilepsy; extracellular signal-regulated kinase (ERK); growth hormone secretagogues; hippocampus.

Figure 1

Behavioral response to repeated 6-Hz corneal stimulation in mice treated with saline or EP-80317. Seizures were evaluated for severity and duration. Severity was measured as the percentage of mice developing generalized tonic-clonic seizures associated with loss of posture (A). Note that loss of posture occurred in a progressively higher percentage of saline-treated mice (^#P < 0.05 vs. session 1 in the same treatment group, Fisher’s exact test; *P < 0.05 vs. saline-treated mice in sessions 2 and 3). The seizure duration (B–D) was evaluated separately for convulsions (C, motor component) and stunned posture (D, non-motor component), or as overall duration (B, including both components). Note that the overall duration decreased progressively after each session in both groups (^##P < 0.01, session 3 vs. 1; Fisher’s least significant difference (LSD) test). Interestingly, a significantly lower overall duration was initially found in mice treated with EP-80317 (*P < 0.05 vs. saline-treated mice in session 1; Fisher’s LSD test). When considering only the motor component (C), this was reduced by EP-80317 in the first two sessions (*P < 0.05). Concerning the non-motor component (D), a significant decrease was found in saline-treated mice (^##P < 0.01, session 2 and 3 vs. session 1). In mice treated with EP-80317 group, the non-motor component did not change in the various sessions and it was initially shorter than in saline-treated mice (**P < 0.01, session 1).

Figure 2

Electrocorticographic (ECoG) activity during seizures induced by 6-Hz corneal stimulation. EcoG traces obtained from frontal cortex of one out of three representative mice. Note that the trace clearly shows two components: (i) the ictal event corresponding to the motor component of the seizure; and (ii) the post-ictal event, characterized by flattening of basal activity corresponding to the non-motor component of seizures. Traces were respectively taken from a saline-treated mice (A) or an animal treated with EP-80317 (B). No quantification is provided because of the low number of animals per group (n = 3). RFC, right frontal cortex.

Figure 3

FosB/ΔFosB immunoreactivity in hippocampal regions of mice treated with saline or EP-80317 and exposed to different sessions of 6-Hz corneal stimulation. In (A), FosB/ΔFosB immunoreactivity is illustrated in CA1, CA3, and dentate gyrus (DG), in a representative unstimulated control (ctrl) mouse. Besides, FosB/ΔFosB immunoreactivity is also shown in mice exposed to one or three different sessions of 6-Hz corneal stimulation, and pretreated with saline (sal) or EP-80317 (ep). Immunoreactivity was measured and results are illustrated in (B–D). Note that FosB/ΔFosB levels were significantly increased in CA1 (B), CA3 (C), and DG (D) after the first session, in saline-treated mice only (°°P < 0.01, °°°P < 0.001 vs. controls; Fisher’s least significant difference test). In EP-80317-treated mice, FosB/ΔFosB levels were instead maintained at basal values and were significantly different from saline-treated mice (***P < 0.001 vs. sal-1). Note also that FosB/ΔFosB levels were significantly reduced in the third session of saline-treated mice (^##P < 0.01, ^###P < 0.001 vs. sal-1). Scale bar, 50 μm.

Figure 4

Changes in phosphorylated extracellular signal-regulated kinases 1/2 (p-ERK1/2) immunoreactivity in hippocampal regions of mice treated with EP-80317 and exposed to different sessions of 6-Hz corneal stimulation. In (A), p-ERK1/2 immunoreactivity is illustrated in CA1, CA3, and DG, in a representative unstimulated control (ctrl) mouse. Moreover, p-ERK1/2 immunoreactivity is also shown in mice exposed to one or three different sessions of 6-Hz corneal stimulation and pretreated with saline (sal) or EP-80317 (ep). Immunoreactivity was measured and results are illustrated in (B–D). Note that p-ERK1/2 expression was initially increased in CA1 (°P < 0.05 vs. controls; Fisher’s least significant difference test), both in EP-80317 and saline-treated mice. After the third seizure, immunopositive cells increased also in CA3 (C) and DG (D) (°°P < 0.01, °°°P < 0.001 vs. controls) and were higher than in the initial session (^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. session 1 of the respectively considered group of treatment). However, the changes observed in mice treated with EP-80317 were less pronounced than in saline-treated mice (***P < 0.001 vs. sal-3). Scale bar, 50 μm.

Figure 5

Double immunofluorescence confocal laser scanning micrographs of mice exposed to repeated 6-Hz corneal stimulation. Photomicrographs illustrating co-labeling with phosphorylated extracellular signal-regulated kinases 1/2 (p-ERK1/2) (green) and FosB/ΔFosB (red) in CA1 of representative mouse for each group of treatment. Double-label immunofluorescence revealed the coexpression of p-ERK1/2 and FosB/ΔFosB in the experimental groups. Scale bar, 75 μm.