Exacerbation of epilepsy by astrocyte alkalization and gap junction uncoupling.

概要

Epilepsy is a common neuronal disorder characterized by hyperexcitability and progressive seizures. It is known that epilepsy become more severe with each incident of seizures. The amount of plastic changes that occur at the very early stage of epilepsy may be a prognosis of epileptogenesis. However, such plasticity has not been thoroughly addressed. Describing the mechanisms of the initial response and the subsequent cascading events would be useful for devising a new preventative medicine for the suppression of epileptogenesis. Here, I demonstrated that acute plastic changes of the astrocytic network occur in response to the first-time exposure to an epileptiform activity.

　Astrocytes, a major class of the macroglial cells, have a strong control over neuronal excitability and the mode of information processing. This control is accomplished by adjusting the levels of various ions in the extracellular space. The network of astrocytes connected via gap junctions allows a wider or more confined distribution of these ions depending on the open probability of the gap junctions. K+ clearance relies on the K+ uptake by astrocytes and the subsequent diffusion of K+ through the astrocyte network. When astrocytes become uncoupled, K+ clearance becomes hindered. Accumulation of extracellular K+ leads to hyperexcitability of neurons, which may further exacerbate seizures.

　The aim of this study was to understand the cellular mechanisms leading to the exacerbation of epilepsy. To this end, I employed electrophysiology and extra- and intra-cellular ion concentration recording techniques to analyze the contribution of astrocyte gap junction coupling in acute mouse hippocampal slices.

　It is possible that only a brief seizure experience could be enough of a trigger to induce plastic changes in the brain, leading to a state prone to more severe hyperactivity. To create such short-term plasticity model of exacerbation of epilepsy, acute hippocampal slice was exposed to Mg2+-free superfusate containing a GABAA receptor antagonist (picrotoxin), which can induce epileptiform activity. Current clamp recordings from pyramidal cells in hippocampal CA1 confirmed the epileptiform-like activity within 15 minutes of the perfusion.

　Extracellular K+ concentrations were recorded with K+-selective microelectrodes. K+ transients in response to glutamate application were measured. In slices that experienced epileptiform activity, duration of the K+ clearance became significantly prolonged. This confirms that epileptiform activity leads to impairment of K+ clearance.

　To reveal the mechanisms underlying the hindrance of K+ clearance, the degree of gap junction coupling between astrocytes was evaluated. I performed Na+ imaging with a fluorescent indicator (SBFI) to track the inter- cellular diffusion of small cations in the astrocytic syncytium via gap junctions. Diffusion via gap junctions rapidly became restricted after epileptiform activity.

　To further explore the mechanisms underlying gap junction uncoupling, pH imaging was performed using a transgenic mouse with astrocyte specific expression of a pH sensor (Lck-E2GFP). I confirmed that astrocytes react to epileptiform activity with intracellular alkalization via activation of Na+/HCO - co-transporter (NBC). By the pharmacological blockade of NBC, intracellular alkalization, gap junction uncoupling, and exacerbation of hyperactivity was prevented in vitro. The anti-epileptic effect of the NBC blocker was also demonstrated in an in vivo mouse model of progressive epilepsy.

　These data thus demonstrate a functional plasticity of astrocytes in response to short-term hyperactivity of neurons, which apparently leads to exacerbation of epilepsy. By uncovering the mechanisms underlying the plasticity of astrocyte gap junctions, I was able to propose a possible target for suppressing epileptogenesis at the very early stage.

　This finding shows the powerful effects of the astrocyte state on the function of neuronal circuits. Such astrocyte plasticity may occur in both physiological and pathophysiological situations and contribute to the outstanding dynamism of the brain function.