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, 60 (1), 83-95

Local Disruption of Glial Adenosine Homeostasis in Mice Associates With Focal Electrographic Seizures: A First Step in Epileptogenesis?

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Local Disruption of Glial Adenosine Homeostasis in Mice Associates With Focal Electrographic Seizures: A First Step in Epileptogenesis?

Tianfu Li et al. Glia.

Abstract

Astrogliosis and associated dysfunction of adenosine homeostasis are pathological hallmarks of the epileptic brain and thought to contribute to seizure generation in epilepsy. The authors hypothesized that astrogliosis-an early component of the epileptogenic cascade-might be linked to focal seizure onset. To isolate the contribution of astrogliosis to ictogenesis from other pathological events involved in epilepsy, the authors used a minimalistic model of epileptogenesis in mice, based on a focal onset status epilepticus triggered by intra-amygdaloid injection of kainic acid. The authors demonstrate acute neuronal cell loss restricted to the injected amygdala and ipsilateral CA3, followed 3 weeks later by focal astrogliosis and overexpression of the adenosine-metabolizing enzyme adenosine kinase (ADK). Using synchronous electroencephalographic recordings from multiple depth electrodes, the authors identify the KA-injected amygdala and ipsilateral CA3 as two independent foci for the initiation of non-synchronized electrographic subclinical seizures. Importantly, seizures remained focal and restricted to areas of ADK overexpression. However, after systemic application of a non-convulsive dose of an adenosine A(1) -receptor antagonist, seizures in amygdala and CA3 immediately synchronized and spread throughout the cortex, leading to convulsive seizures. This focal seizure phenotype remained stable over at least several weeks. We conclude that astrogliosis via disruption of adenosine homeostasis per se and in the absence of any other overt pathology, is associated with the emergence of spontaneous recurrent subclinical seizures, which remain stable over space and time. A secondary event, here mimicked by brain-wide disruption of adenosine signaling, is likely required to turn pre-existing subclinical seizures into a clinical seizure phenotype.

Figures

Figure 1
Figure 1
Intraamygdaloid injection of KA induces status epilepticus. The upper trace is a representative 60 minute cortical EEG recording that includes the 30 minute period of status epilepticus terminated by lorazepam injection. The lower trace is a high resolution 10 sec segment obtained from the upper trace (demarcated by orange highlight) that is an example of type IV seizure activity.
Figure 2
Figure 2
Intraamygdaloid KA injection induces acute cell death within the ipsilateral amygdala. A–D, Representative images of Nissl staining in the contra- (A,C) and ipsilateral (B,D) amygdala 24 hours after intraamygdaloid KA injection. A,B, Disrupted Nissl staining in the ipsilateral amygdala (B, circle) and the corresponding unaffected contralateral region (A, circle). C,D, Higher magnification images of the Nissl stained regions demarcated by circles in panels A and B, respectively. E,F, TUNEL labeling of sections adjacent to panels A and B. Scale bars: 300 μm (AD), 75 μm (E–F).
Figure 3
Figure 3
A single focal intraamygdaloid KA injection causes acute cell death that is restricted to the CA3. A–D, Representative Nissl stained sections showing CA3 selective damage (circles) throughout the rostral-caudal extent of the hippocampus 24 hours post intraamygdaloid KA-injection. E–N, Representative images of TUNEL labeled cells (E–I) and DAPI stained nuclei (J–N) that correspond to the respective areas demarcated by circles in panels A–D. Insets depict TUNEL labeling (F) and DAPI staining (K, arrows) of condensed nuclei. Scale bars: 300 μm (A–D), 75 μm (E–N).
Figure 4
Figure 4
Intraamygdaloid KA injection causes chronic injury associated with astrogliosis and ADK overexpression in the ipsilateral amygdala and CA3. A1,2;B1,2, Representative Nissl staining of the injured (A2, B2; circles) and corresponding non-injured (A1, B1; circles) amygdala (A1,2) and CA3 (B1,2) 3 weeks after KA injection. A3-8;B3-8, Representative images of GFAP (A3,4;B3,4) and ADK (A5-8;B5-8) immunoreactive material in the amygdala (A3-8) and CA3 (B3-8). Increased GFAP (A4,B4; circles) and ADK (A6,B6; circles) immunoreactive material is only present in the ipsilateral injury site and correspond to disrupted Nissl staining. A7,8;B7,8, High magnification images obtained from the contralateral amygdala (A7) and CA3 (B7) and within the area demarcated by circles in panels A6 and B6 for the ipsilateral amygdala (A8) and CA3 (B8), respectively. Scale bars: 300 μm (A1-6;B1-6), 37.5 μm (A7,8;B7,8).
Figure 5
Figure 5
Chronic injury from intraamygdaloid KA injection causes brain region-specific changes in ADK expression levels. Low magnification image of ADK immunoreactive material in a coronal brain section from a KA injected mouse three weeks after SE. The circles demarcate the ipsilateral amygdala and CA3 regions which have increased ADK expression in comparison to the corresponding contralateral regions. Scale bar: 500 μm.
Figure 6
Figure 6
ADK upregulation colocalizes with astrogliosis in the injured amygdala and CA3. A1-4;B1-4, Representative images of GFAP (A1,2;B1,2, green) and ADK (A3,4;B3,4, red) double labeled sections that were acquired from within the amygdala and CA3 three weeks after intraamygdaloid KA injection. The ipsilateral amygdala (A2,4) and CA3 (B2,4) have increased GFAP (green) and ADK (red) immunoreactive material compared to the contralateral hemispheres (A1,3;B1,3). A5,6;B5,6, Merged fluorescence image of GFAP and ADK immunoreactive material. The increased levels of ADK immunoreactive material (red) colocalizes with reactive astrocytes (GFAP, green) within the ipsilateral amygdala (A6) and CA3 (B6). Scale bars: 37.5 μm (A1-6;B1-6).
Figure 7
Figure 7
Focal spontaneous seizures develop in the amygdala and CA3 three weeks after intraamygdaloid KA injection. A, High resolution EEG trace of the amygdala-originating spontaneous focal electrographic seizure depicted in panel B1. B, Recordings three weeks after KA injection from bipolar electrodes implanted into the amygdala (Amg) and CA3 and from a monopolar electrode implanted in the cortex (CTX). Note that spontaneous electrographic seizures are focal, but alternate in origin between the Amg (B1) and CA3 (B2). DPCPX injection (1 mg/kg in 20% DMSO/saline, i.p) synchronizes electrographic seizure activity in the Amg and CA3 with subsequent generalization to the CXT (right). C, Recordings from the Amg, CA3 and CTX in control saline-injected mice. Note that there was no response to DPCPX (right). Scale bars: 1 second (A); 10 seconds (B,C).
Figure 8
Figure 8
Spontaneous electrographic seizures in the hippocampus are confined to the CA3 subregion. A, High resolution EEG trace of the CA3-originating spontaneous focal electrographic seizure depicted in panel B. B, Recordings three weeks after KA injection from bipolar electrodes implanted into the CA1/CA3/dentate gyrus (DG) and monopolar electrode in the cortex (CTX). DPCPX injection (1 mg/kg, i.p) generalizes electrographic seizure activity throughout the hippocampus and into the CTX (right). C, Recordings from the CA3/CA1/DG and CTX in control saline-injected mice. Note that there wasno response to DPCPX (right). Scale bars: 1 second (A); 10 seconds (B,C).
Figure 9
Figure 9
Hippocampal damage from intraamygdaloid KA is persistent. A–D, ADK immunoreactive material in the contralateral (A,C) and ipsilateral (B,D) CA3 two months after KA injection. E, Recordings from mice implanted with bipolar electrodes into the CA3/CA1/dentate gyrus (DG) and reference electrode into the cortex (CTX) two months after KA injection. Note that spontaneous electrographic seizure activity is localized to the CA3 subregion. Scale bars: 300 μm (A, B), 37.5 μm (C,D).

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