Brain injury may result in the development of epilepsy, one of the most common neurological disorders. We previously demonstrated that albumin is critical in the generation of epilepsy after blood-brain barrier (BBB) compromise. Here, we identify TGF-␤ pathway activation as the underlying mechanism. We demonstrate that direct activation of the TGF-␤ pathway by TGF-␤1 results in epileptiform activity similar to that after exposure to albumin. Coimmunoprecipitation revealed binding of albumin to TGF-␤ receptor II, and Smad2 phosphorylation confirmed downstream activation of this pathway. Transcriptome profiling demonstrated similar expression patterns after BBB breakdown, albumin, and TGF-␤1 exposure, including modulation of genes associated with the TGF-␤ pathway, early astrocytic activation, inflammation, and reduced inhibitory transmission. Importantly, TGF-␤ pathway blockers suppressed most albumininduced transcriptional changes and prevented the generation of epileptiform activity. Our present data identifies the TGF-␤ pathway as a novel putative epileptogenic signaling cascade and therapeutic target for the prevention of injury-induced epilepsy.

Focal epilepsy often develops following traumatic, ischemic, or infectious brain injury. While the electrical activity of the epileptic brain is well characterized, the mechanisms underlying epileptogenesis are poorly understood. We have recently shown that in the rat neocortex, long-lasting breakdown of the blood-brain barrier (BBB) or direct exposure of the neocortex to serum-derived albumin leads to rapid upregulation of the astrocytic marker GFAP (glial fibrillary acidic protein), followed by delayed (within 4 -7 d) development of an epileptic focus. We investigated the role of astrocytes in epileptogenesis in the BBB-breakdown and albumin models of epileptogenesis. We found similar, robust changes in astrocytic gene expression in the neocortex within hours following treatment with deoxycholic acid (BBB breakdown) or albumin. These changes predict reduced clearance capacity for both extracellular glutamate and potassium. Electrophysiological recordings in vitro confirmed the reduced clearance of activity-dependent accumulation of both potassium and glutamate 24 h following exposure to albumin. We used a NEURON model to simulate the consequences of reduced astrocytic uptake of potassium and glutamate on EPSPs. The model predicted that the accumulation of glutamate is associated with frequency-dependent (Ͼ100 Hz) decreased facilitation of EPSPs, while potassium accumulation leads to frequency-dependent (10 -50 Hz) and NMDAdependent synaptic facilitation. In vitro electrophysiological recordings during epileptogenesis confirmed frequency-dependent synaptic facilitation leading to seizure-like activity. Our data indicate a transcription-mediated astrocytic transformation early during epileptogenesis. We suggest that the resulting reduction in the clearance of extracellular potassium underlies frequencydependent neuronal hyperexcitability and network synchronization.

It is widely believed that, in cortical pyramidal cells, action potentials (APs) initiate in the distal portion of axon initial segment (AIS) because that is where Na + channel density is highest. To investigate the relationship between the density of Na + channels and the spatiotemporal pattern of AP initiation, we simultaneously recorded Na + flux and action currents along the proximal axonal length. We found that functional Na + channel density is approximately four times lower in the AP trigger zone than in the middle of the AIS, where it is highest. Computational analysis of AP initiation revealed a paradoxical mismatch between the AP threshold and Na + channel density, which could be explained by the lopsided capacitive load imposed on the proximal end of the AIS by the somatodendritic compartment. Favorable conditions for AP initiation are therefore achieved in the distal AIS portion, close to the edge of myelin, where the current source-load ratio is highest. Our findings suggest that cable properties play a central role in determining where the AP starts, such that small plastic changes in the local AIS Na + channel density could have a large influence on neuronal excitability as a whole.neocortex | pyramidal neuron | sodium imaging I n cortical pyramidal cells, as in many CNS neurons, action potentials (APs) generally initiate in the axon initial segment (AIS) (refs. 1-4; reviewed in ref. 5), the proximal part of the axon where the neuronal membrane is not covered with a myelin sheath, and which possesses a distinctive, specialized assembly of voltage-gated channels and associated proteins (6). Because of the pivotal role that the AIS plays in transformation of synaptic input into AP output, precise characterization of its excitable properties is essential for a complete understanding of the cellular mechanisms that underlie operation of cortical neurons and networks. Early theoretical studies proposed two mechanisms to explain preferential AIS AP initiation (7, 8): (i) less current is required to depolarize the AIS membrane to threshold because it is electrically isolated from the neighboring neuronal compartments, and (ii) the depolarizing current density is higher in the AIS than in neighboring compartments, allowing the AIS to overcome their electric load. These two mechanisms are not mutually exclusive, but the technical difficulties that hinder precise measurements in thin neuronal processes have made it difficult to elucidate their relative importance for AP initiation.During the past decade, the isolation hypothesis has been addressed by only a few studies (9), and values of the relevant resistances and capacitances are only approximations. By contrast, the findings by many groups that Na + channel density is relatively high in the proximal axon have focused most attention on the higher current hypothesis, although there remains some controversy as to what extent the density of functional Na + channels is greater in the AIS than in the soma (refs. 10-14; reviewed in ref. 15). The role of high ...

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TGF-β1 is a master cytokine in immune regulation, orchestrating both pro- and anti-inflammatory reactions. Recent studies show that whereas TGF-β1 induces a quiescent microglia phenotype, it plays a pathogenic role in the neurovascular unit and triggers neuronal hyperexcitability and epileptogenesis. In this study, we show that, in primary glial cultures, TGF-β signaling induces rapid upregulation of the cytokine IL-6 in astrocytes, but not in microglia, via enhanced expression, phosphorylation, and nuclear translocation of SMAD2/3. Electrophysiological recordings show that administration of IL-6 increases cortical excitability, culminating in epileptiform discharges in vitro and spontaneous seizures in C57BL/6 mice. Intracellular recordings from layer V pyramidal cells in neocortical slices obtained from IL-6–treated mice show that during epileptogenesis, the cells respond to repetitive orthodromic activation with prolonged after-depolarization with no apparent changes in intrinsic membrane properties. Notably, TGF-β1–induced IL-6 upregulation occurs in brains of FVB/N but not in brains of C57BL/6 mice. Overall, our data suggest that TGF-β signaling in the brain can cause astrocyte activation whereby IL-6 upregulation results in dysregulation of astrocyte–neuronal interactions and neuronal hyperexcitability. Whereas IL-6 is epileptogenic in C57BL/6 mice, its upregulation by TGF-β1 is more profound in FVB/N mice characterized as a relatively more susceptible strain to seizure-induced cell death.