Differential Expression of the Metabotropic P2Y Receptor Family in the Cortex Following Status Epilepticus and Neuroprotection via P2Y1 Antagonism in Mice

Abstract

Purinergic signaling via P2 receptors is now widely accepted to play a critical role during increased states of hyperexcitability and seizure-induced pathology. In the setting of seizures and epilepsy, most attention has been paid to investigating the fast-acting ATP-gated P2X receptor family. More recent evidence has now also provided compelling evidence of an involvement of the slower-acting P2Y receptor family during seizures. This includes data demonstrating expression changes of P2Y receptors in the hippocampus following acute seizures and during epilepsy and anticonvulsive properties of P2Y-targeting drugs; in particular drugs targeting the P2Y₁ subtype. Seizures, however, also involve damage to extra-hippocampal brain regions such as the cortex, which is thought to contribute to the epileptic phenotype. To analyze expressional changes of the P2Y receptor family in the cortex following status epilepticus and to determine the impact of drugs interfering with P2Y₁ signaling on cortical damage, we used a unilateral mouse model of intraamygdala kainic acid-induced status epilepticus. Analysis of cortical tissue showed that status epilepticus leads to a global up-regulation of the P2Y receptor family in the cortex including P2Y₁, P2Y₂, P2Y₄, and P2Y₆, with the P2Y₁ and P2Y₄ receptor subtypes showing the strongest increase. Supporting a detrimental role of P2Y₁ activation during status epilepticus, treatment with the P2Y₁ agonist MRS2365 exacerbated high frequency high amplitude spiking, synonymous with injury-causing electrographic activity, and treatment with the P2Y₁ antagonists MRS2500 protected against seizure-induced cortical damage. Suggesting P2Y₁-mediated effects are predominantly due to increased microglia activation, treatment with the broad-spectrum anti-inflammatory drug minocycline abolished the observed neuroprotective effects of P2Y₁ antagonism. In conclusion, our results further support a role for P2Y₁-mediated signaling during seizure generation and seizure-induced neurodegeneration, suggesting P2Y₁-targeting therapies as novel treatment for drug-refractory status epilepticus.

Keywords: adenosine triphosphate; cortex; metabotropic P2 receptor family; neurodegeneration; purinergic signaling; status epilepticus.

Figure 1

Expression profiling of the P2Y receptor family in the cortex following status epilepticus. (A) Photomicrograph (20× lens) showing neuronal damage 24 h following intraamygdala KA-induced status epilepticus in the ipsilateral hippocampus and cortex. Scale bar = 100 μm. (B) Representative Western blot (n = 1 per lane) and corresponding graph showing increased c-Fos expression in the ipsilateral cortex post-status epilepticus (n = 4 per group). (C) Representative Western blots (n = 1 per lane) and corresponding graphs showing the expression of the different P2Y receptor family members P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₂, P2Y₁₃, and P2Y₁₄ in cortical tissue following status epilepticus. Of note, while P2Y₁, P2Y₂, P2Y₄, P2Y₆ are significantly increased post-status epilepticus, no changes could be observed for the remaining P2Y receptors (n = 6 per group). *p < 0.05, **p < 0.01.

Figure 2

Cell-specific expression of P2Y₁ post-status epilepticus. (A) Photomicrographs (40× lens) showing co-localization of P2Y₁ (green) with neuronal marker NeuN in cortical tissue (layer V–VI) in control injected-vehicle mice and following status epilepticus (SE) (white arrows). Scale bar = 10 µm (B) Co-localization of P2Y₁ (green) with microglia marker Iba1 (red) 24 h following status epilepticus (SE) in the cortex (white arrows). Scale bar = 10 μm. (C, D) No co-localization of P2Y₁ (green) with the astrocyte markers GFAP and S100β (red). Scale bar = 10 μm. (E) Graph showing strong increase in P2Y₁-positive microglia 24 h following status epilepticus (n = 3 per group). (F) Enlarged images, outlined by white box, showing co-localization of P2Y₁ (green) on neurons (red) and microglia (red) 24 h post-status epilepticus (SE). Scale bar = 10 μm. (G) Specificity of P2Y₁-detecting antibody was confirmed using tissue from P2Y₁ knockout mice (KO) 8 h post-status epilepticus. Scale bar = 10 μm. Images are representative image from three animals per experiment. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

P2Y₁ antagonism decreases high frequency high amplitude spiking during status epilepticus. (A) Representative EEG traces recorded from the cortex from the time-point of intraamygdala kainic acid (KA) injection until 60 min post-lorazepam (Lz) of mice treated with vehicle (Veh), P2Y₁ agonist MRS2365 (MRS23), and P2Y₁ antagonist MRS2500 (MRS25). Treatment with P2Y₁-targeting drugs was administered 15 min post-intraamygdala KA injection via i.c.v. Lorazepam (Lz) was administered 40 min following KA injection via i.p. (B) Representative EEG traces showing examples of high frequency and high amplitude (HFHA) spiking taken during the 30 min recording period following drug treatment (see arrows). Mice treated with the P2Y₁ agonist MRS23 showed an increase in the duration of HFHA spiking while mice treated with the P2Y₁ antagonists MRS25 showed a decrease in the duration of HFHA spiking during status epilepticus (n = 7 Veh, 9 MRS23, and 8 MRS25). (C) Representative EEG traces and graph showing slightly increased duration of HFHA spiking during status epilepticus in mice treated with minocycline and P2Y₁ antagonists MRS25 (n = 4 per group). *p < 0.05, **p < 0.01.

Figure 4

Decreased status epilepticus-induced cortical damage via P2Y₁ antagonism. (A) Representative images (20× lens) and corresponding graph showing a decrease in neuronal damage in the cortex 24 h post-status epilepticus in mice treated with the P2Y₁ antagonist MRS25 (n = 7 Veh, 9 MRS23, and 8 MRS25). (B) Representative images (20x lens) and corresponding graph showing slightly more Fluoro-Jade B (FjB)-positive cells in ipsilateral cortex 24 h post-status epilepticus in mice treated with both minocycline and the P2Y1 antagonist MRS25 (n = 4/group). **p < 0.01.