Experimental febrile seizures impair interastrocytic gap junction coupling in juvenile mice

Abstract

Prolonged and focal febrile seizures (FSs) have been associated with the development of temporal lobe epilepsy (TLE), although the underlying mechanism and the contribution of predisposing risk factors are still poorly understood. Using a kainate model of TLE, we previously provided strong evidence that interruption of astrocyte gap junction-mediated intercellular communication represents a crucial event in epileptogenesis. To elucidate this aspect further, we induced seizures in immature mice by hyperthermia (HT) to study the consequences of FSs on the hippocampal astrocytic network. Changes in interastrocytic coupling were assessed by tracer diffusion studies in acute slices from mice 5 days after experimental FS induction. The results reveal that HT-induced FSs cause a pronounced reduction of astrocyte gap junctional coupling in the hippocampus by more than 50%. Western blot analysis indicated that reduced connexin43 protein expression and/or changes in the phosphorylation status account for this astrocyte dysfunction. Remarkably, uncoupling occurred in the absence of neuronal death and reactive gliosis. These data provide a mechanistic link between FSs and the subsequent development of TLE and further strengthen the emerging view that astrocytes have a central role in the pathogenesis of this disorder. © 2016 Wiley Periodicals, Inc.

Commonalities in epileptogenic processes from different acute brain insults: Do they translate?

“…Reduced glial K + conductances and loss of astrocyte gap junction coupling compromise the clearance of extracellular K + and impair K + homeostasis. Loss of coupling is mediated by proinflammatory cytokines and precedes alterations in neurons and K + clearance, suggesting a causal role of dysfunctional K + buffering by astrocytes in the etiology of different epilepsies . Reduced astrocyte coupling was also reported after acute CVA in situ .…”

“…A common pathological hallmark in the sequence of events converting a normal brain into an epileptic brain after an acquired insult is microglial activation and subsequent astrogliosis, which result from inflammatory processes . Astrocytes regulate and amplify immune‐mediated mechanisms implicated in epileptogenesis . Evidence from experimental epileptogenesis studies and from studies of established human epilepsy shows that astrocytes undergo functional changes including dysregulation of astroglial K ir and gap junction channels, which together alter glioneuronal communication and, by impairing uptake and redistribution of extracellular K + accumulated during neuronal activity, can contribute to or cause seizures .…”

“…Astrocytes regulate and amplify immune‐mediated mechanisms implicated in epileptogenesis . Evidence from experimental epileptogenesis studies and from studies of established human epilepsy shows that astrocytes undergo functional changes including dysregulation of astroglial K ir and gap junction channels, which together alter glioneuronal communication and, by impairing uptake and redistribution of extracellular K + accumulated during neuronal activity, can contribute to or cause seizures . Downregulation of astrocytic K ir channels appears to be a common response to brain injury and has been observed after TBI, CVA, BBB damage, entorhinal cortex lesions, and freeze‐lesion–induced cortical dysplasia, and in association with experimentally induced posttraumatic epilepsy .…”

Subcellular reorganization and altered phosphorylation of the astrocytic gap junction protein connexin43 in human and experimental temporal lobe epilepsy

“…Thirty µg of these protein lysates were subjected to SDS PAGE followed by Western blotting. All blotted membranes were blocked with 5% nonfat milk in TBST for 1 hr followed by overnight incubation with primary antibodies (custom‐made rabbit anti‐Cx43 antibody, dilution 1:2500 (Khan et al, ); rabbit anti‐Cx30 antibody, Invitrogen/Thermoscientific, catalogue number 71–2200, dilution 1:250; rabbit anti‐E‐cadherin antibody, Abcam, ab15148, catalogue number: 71–2200, dilution 1:1000) in 2.5% milk. The next day, after 3 times washings in TBST, the membranes were probed with ECL™ Rabbit IgG, HRP‐linked F(ab′) 2 fragment (Amersham, catalogue number: NA 9340, dilution 1:5000) in 2.5% milk for 1 hr.…”

Neuron–glia interaction in epilepsy

“…Juvenile febrile seizures (FS) are suspected of representing predisposing events in triggering human TLE. According to the original paper in this issue by Khan et al () analyzing a hyperthermia mouse model of juvenile FS, impaired gap junction coupling between astrocytes might play a key role in epileptogenesis. Based on a previous study revealing cytokines as a cause of uncoupling in TLE (Bedner et al, ), it is likely that microglial activation contributed to uncoupling in the FS model, which remarkably occurred in the absence of neuronal death and astrogliosis.…”