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MANUSCRIPT ID

• Title

Enhancement of GABAA-current run-down in the hippocampus occurs at the first spontaneous seizure in a model of temporal lobe epilepsy

• Reference

Manuela Mazzuferia, Eleonora Palmac, Katiuscia Martinelloe, Francesca Maiolinod, Cristina Rosetic, Sergio Fucilec, Paolo F. Fabenef, Federica Schiof, Michele Pellitterif, Guenther Sperkg, Ricardo Miledih, Fabrizio Eusebic, and Michele Simonatoa, Proc Natl Acad Sci U S A. 2010 Feb 16;107(7):3180-5. Epub 2010 Jan 26.

• Abstract

Refractory temporal lobe epilepsy (TLE) is associated with a dysfunction of inhibitory signaling mediated by GABAA receptors. In particular, the use-dependent decrease (run-down) of the currents (IGABA) evoked by the repetitive activation of GABAA receptors is markedly enhanced in hippocampal and cortical neurons of TLE patients. Understanding the role of IGABA run-down in the disease, and its mechanisms, may allow development of medical alternatives to surgical resection, but such mechanistic insights are difficult to pursue in surgical human tissue. Therefore, we have used an animal model (pilocarpine-treated rats) to identify when and where the increase in IGABA run-down occurs in the natural history of epilepsy. We found: (i) that the increased run-down occurs in the hippocampus at the time of the first spontaneous seizure (i.e., when the diagnosis of epilepsy is made), and then extends to the neocortex and remains constant in the course of the disease; (ii) that the phenomenon is strictly correlated with the occurrence of spontaneous seizures, because it is not observed in animals that do not become epileptic. Furthermore, initial exploration of the molecular mechanism disclosed a relative increase in α4-, relative to α1-containing GABAA receptors, occurring at the same time when the increased run-down appears, suggesting that alterations in the molecular composition of the GABA receptors may be responsible for the occurrence of the increased run-down. These observations disclose research opportunities in the field of epileptogenesis that may lead to a better understanding of the mechanism whereby a previously normal tissue becomes epileptic.

• Keywords

GABAA receptor | pilocarpine rat | Xenopus oocytes

• Input author

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MANUSCRIPT DETAILS

• Introduction/Aims

Nearly one third of patients with temporal lobe epilepsy (TLE) are refractory to treatment with current anti-epileptic drugs (AEDs), and must resort to surgical resection of the epileptic focus for relief. In order to design better treatments for these patients, it would be helpful to better understand the mechanisms involved in the process of epileptogenesis. An increase toward hyperexcitability due to a reduction in inhibition has been a generally proposed mechanism. More specifically, GABA current run-down has been shown to occur in epileptic tissue ablated during epilepsy surgery. However, it is unclear whether the run-down observed in human epileptic tissue occurred due to the seizures or was part of the epileptogenic process.

The aim of this paper was to determine the timing of this mechanism in the course of the disease progression.

• Method:

A commonly used model of temporal lobe epileptogenesis, the rat pilocarpine seizure model, was studied to determine the timing of GABA current run-down during the epileptogenic process—in the so called “silent period” between the epileptogenic insult (pilocarpine-induced status epilepticus) and the first clinically detectable spontaneous seizure.

Video-EEG monitoring was used to determine the timing of the first spontaneous recurrent seizure (SRS) thus the onset of clinical epilepsy.

Animals were sacrificed at 24 hours (acute time point), 5 days (latency), following first spontaneous seizure (~9 days), 2 wks following first seizure (chronic phase), or 2 months following the first seizure (late chronic phase). Comparisons were made between tissue from epileptic animals at these time points and naïve tissue. A second control group was composed of “resistant” animals, which did not become epileptic following the epileptogenic insult.

Tissue was either 1) homogenized into membrane vesicles from cortex or hippocampal tissue and injected into xenopus oocytes or 2) prepared into coronal sections for immunohistochemical and electrophysiological analyses. Whole cell patch clamp was used to measure GABA current run-down in response to repetitive exogenously released GABA puffs on oocytes or slices preparations.

• Results

Figure 1. (A) Schematic diagram of the in vivo experiments. The time points of analysis are indicated by red arrows. (B) Time course of neurodegeneration in the neocortex. (Ca) Time course of neurodegeneration in the whole hippocampus. (Cb) and (Cc) Representative images of the effect of pilocarpine SE on degeneration in the hippocampus. (Mazzuferi et al. 2010)

Figure 2. Neuronal loss and astrocytosis at various time points after pilocarpine-induced SE (Mazzuferi et al. 2010)

Figure 3. IGABA run-down from oocytes injected with membranes prepared from rats killed at various time points after pilocarpine-induced SE (Mazzuferi et al. 2010)

1) Both astrocytosis and neuron loss peak during the latency period prior to the onset of the first seizure. The resistant animal showed no astrocytosis or neuron loss at the two-month time point compared to control.

2) Hippocampus: GABAa current run-down & increase in alpha4 with onset of SRS

3) Cortex: Run-down & increase in alpha4 subunit occur together at week two.

4) No change in ratio of alpha1/alpha4 subunitcontaining GABA receptors among resistant group (nice control for SE, pilocarpine and diazepam).

5) GABA current run-down may make tissue hyper-excitable.

Figure 4. Different IGABA run-down upon repetitive stimulation in CA3 pyr- amidal neurons in hippocampal slices from control rats, pilocarpine-treated rats in acute phase (24 h after SE) and chronically epileptic rats (Mazzuferi et al. 2010)

Figure 5. Relative representation of the alpha1 and alpha4 subunits in CA3, at various time points after pilocarpine-induced SE (Mazzuferi et al. 2010)

• Discussion

The authors of this paper provide good evidence in support of the notion that changes in GABA subunit composition lead to loss in some inhibitory function, which may lead to hyperexcitability and epilepsy.