Abstract
Astrocyte Control of Synaptic NMDA Receptors Contribute to the Progressive Development of Temporal Lobe Epilepsy.
Clasadonte J, Dong J, Hines DJ, Haydon PG. Proc Natl Acad Sci USA 2013 Oct 22;110(43):17540–17545.
Astrocytes modulate neuronal activity, synaptic transmission, and behavior by releasing chemical transmitters in a process termed gliotransmission. Whether this process impacts epilepsy in vivo is not known. We show that genetic impairment of transmitter release from astrocytes by the expression of a glial dominant negative SNARE domain in mice reduced epileptiform activity in situ, delayed seizure onset after pilocarpine-induced status epilepticus, and attenuated subsequent progressive increase in seizure frequency in vivo. The reduced seizure frequency was accompanied by attenuation of hippocampal damage and behavioral deficits. As the delay in seizure onset and the reduced seizure frequency were mimicked by intracerebroventricular delivery of the NMDA receptor (NMDAR) antagonist D-(-)-2-amino-5-phosphonopentanoate in WT littermates and because dominant-negative SNARE expression leads to a hypofunction of synaptic NMDARs, we conclude that astrocytes modulate epileptogenesis, recurrent spontaneous seizures, and pathophysiological consequences of epilepsy through a pathway involving NMDARs.
Commentary
Historically, epilepsy research has focused on understanding the dysfunction of neuronal cell types in the progression of temporal lobe epilepsy (TLE). More recently, a subset of glial cells—known as astrocytes—have become implicated in the process of epileptogenesis. Following status epilepticus (SE), astrocytes undergo a process termed “reactive gliosis” wherein these cells exhibit dramatic changes in morphology and protein expression (1). These changes are also a hallmark of TLE; however, the functional consequences of this process on disease progression remain unclear (2). Reports have demonstrated that astrocytes begin to increase expression of a variety of receptors including metabotropic glutamate receptors (3) and kainate receptors (4) in SE-induced epilepsy models, suggesting that astrocytes may be responding to changes in extracellular glutamate in the synaptic cleft during seizures. Astrocytes are considered an integral part of the tripartite synapse and have been hypothesized to modulate synaptic transmission via the vesicular release of neurotransmitters including glutamate (5), though this remains a controversial topic within the glial biology field (6). This model suggests that astrocytes could contribute to the excitation of surrounding neurons through a direct mechanism, and thus may offer a new potential therapeutic target in epilepsy.
The greatest challenge faced by those studying the role of astrocytes in epilepsy has been a lack of astrocyte-specific tools to dissect the contribution of these cells to neuronal networks. Fortunately, in the last few years, the increased accessibility of cell type-specific inducible transgenic mice and optogenetic sensors, such as the GCaMP family of proteins, offers hope that exciting breakthroughs lay just beyond the horizon (7). In this vein, a recent study by Clasadonte and colleagues used astrocyte-specific transgenic mice to investigate the role that astrocyte-mediated transmitter release may play in the progression of TLE.
Previously this group developed a line of inducible dominant negative SNARE (dnSNARE) transgenic mice, wherein the vesicular machinery required for the release of transmitters is selectively disrupted in astrocytes (8). In the current study, 2-month-old dnSNARE and WT male littermate mice were administered low-dose injections of pilocarpine and allowed to undergo status epilepticus (SE) for 90 minutes before terminating the SE with diazepam. After a latent period following SE, these mice went on to develop TLE, characterized by spontaneous recurrent seizures. Appropriately, this progressive development of seizures was tracked using long-term video EEG recording. The dnSNARE mice had a longer latency to the development of spontaneous recurrent seizures than did wild-type age-matched controls. Furthermore, during the chronic epilepsy period, the dnSNARE mice had less severe seizures, a slower progression of seizure severity over time, and a reduction in the number of interictal spikes as compared to wild-type controls, suggesting that the loss of vesicular release of signaling molecules in the dnSNARE mice could modify epileptogenesis.
The investigators also looked at behavioral and histological markers associated with TLE 8 months after SE. They assessed locomotor activity using an open field behavior test. Intriguingly, wild-type mice treated with pilocarpine showed less locomotor activity than their dnSNARE counterparts. Clasadonte et al. also quantified the relative number of neurons using an antibody for NeuN. Profound neuronal cell death was associated with SE in the hilar region of the dentate gyrus in WT mice but not in dnSNARE mice suggesting that the dnSNARE mutation is neuroprotective. The authors also investigated reactive gliosis using immunohistochemistry for glial fibrillary acidic protein (GFAP, a protoplasmic astrocytic marker) and found less GFAP expression in the dnSNARE mice after SE compared to controls. These studies pointed to a reduced pathology of brain regions that are normally quite sensitive to SE.
They then turned to an in vitro acute brain slice model of epileptiform activity to investigate changes in synaptic physiology. In brain slices prepared from dnSNARE mice there was a reduced latency to onset of epileptiform activity, and a decrease in the number of ictal-like events compared to WT mice. Furthermore, patch-clamp recordings from CA1 pyramidal neurons 5 months after pilocarpine treatment indicated that NMDA currents were significantly reduced in the dnSNARE mice. Finally, Clasadonte and coauthors found that treatment with D-AP5, an NMDA receptor antagonist, during the latent period of epileptogenesis was sufficient to phenocopy the long-term effects of astrocytic dnSNARE expression, suggesting that astrocytic vesicular glutamate release exerts pro-epileptogenic effects selectively through NMDA receptor activation.
This investigation offered a valuable new perspective on understanding the role of astrocyte-derived chemical transmission on the process of epileptogenesis. A particular strength of the study was the use of astrocyte-specific inducible genetic techniques, as selectively distinguishing astrocyte versus neuronal contributions to networks has been a continuous limitation in other studies attempting to understand astrocyte function in epileptogenesis.
Furthermore, the use of long-term chronic EEG and behavioral monitoring was another considerable strength of the study and provided important relevant data on the various phases of TLE disease progression. One concerning aspect of the experimental design was the lack of electrographic monitoring during pilocarpine-induced SE for all mice in the study. Although behavioral seizures were assessed, this evidence alone is inadequate to conclude that the severity of the SE was the same in both the dnSNARE and their wild-type counterparts. Thus, it cannot be ruled out that the severity of SE was reduced in some of the dnSNARE mice possibly explaining the reductions in seizures, as well as histologic and behavioral differences between the two groups. In addition, analysis of seizures was performed only on 4 to 6 individual mice. Given the variability in number of seizures among the different cohorts, forthcoming studies should include larger sample sizes when assessing behavioral and electrographic seizure measures.
Future directions will hopefully address the selective mechanisms that control glutamate release from astrocytes in wild-type mice as this still remains a contentious topic within the field of astrocyte physiology. In addition, caution should be taken not to conflate phenocopying with the elucidation of a direct mechanism. Furthermore, specific elaboration of the NMDAR-dependent pathway in which astrocyte-selective inhibition of vesicle release is effective in diminishing progression of TLE is warranted. While the approach taken in this study suggests that glutamate released from astrocytes may be contributing to the progression of TLE, the authors did not rule out contributions from other gliotransmitters, such as D-serine, a required co-agonist of the NMDA receptor.
Most excitingly, the study by Clasadonte and colleagues provides evidence suggesting that astrocytes could serve as potential disease-modifying targets in TLE, the Holy Grail for epilepsy researchers. Thus, our goal should be to conduct experiments that provide nuanced information about the mechanistic role of astrocytes during epileptogenesis. With so many new and exciting emerging technologies, obtaining robust data that clarifies and strengthen our models is well within reach.