It has long been recognized that insults to the cerebral cortex, such as trauma, ischaemia or infections, may result in the development of epilepsy, one of the most common neurological disorders. Human and animal studies have suggested that perturbations in neurovascular integrity and breakdown of the blood-brain barrier (BBB) lead to neuronal hypersynchronization and epileptiform activity, but the mechanisms underlying these processes are not known. In this study, we reveal a novel mechanism for epileptogenesis in the injured brain. We used focal neocortical, long-lasting BBB disruption or direct exposure to serum albumin in rats (51 and 13 animals, respectively, and 26 controls) as well as albumin exposure in brain slices in vitro. Most treated slices (72%, n = 189) displayed hypersynchronous propagating epileptiform field potentials when examined 5-49 days after treatment, but only 14% (n = 71) of control slices showed similar responses. We demonstrate that direct brain exposure to serum albumin is associated with albumin uptake into astrocytes, which is mediated by transforming growth factor beta receptors (TGF-betaRs). This uptake is followed by down regulation of inward-rectifying potassium (Kir 4.1) channels in astrocytes, resulting in reduced buffering of extracellular potassium. This, in turn, leads to activity-dependent increased accumulation of extracellular potassium, resulting in facilitated N-methyl-d-aspartate-receptor-mediated neuronal hyperexcitability and eventually epileptiform activity. Blocking TGF-betaR in vivo reduces the likelihood of epileptogenesis in albumin-exposed brains to 29.3% (n = 41 slices, P < 0.05). We propose that the above-described cascade of events following common brain insults leads to brain dysfunction and eventually epilepsy and suggest TGF-betaRs as a possible therapeutic target.

Perturbations in the integrity of the blood-brain barrier have been reported in both humans and animals under numerous pathological conditions. Although the blood-brain barrier prevents the penetration of many blood constituents into the brain extracellular space, the effect of such perturbations on the brain function and their roles in the pathogenesis of cortical diseases are unknown.In this study we established a model for focal disruption of the blood-brain barrier in the rat cortex by direct application of bile salts. Exposure of the cerebral cortex in vivo to bile salts resulted in long-lasting extravasation of serum albumin to the brain extracellular space and was associated with a prominent activation of astrocytes with no inflammatory response or marked cell loss. Using electrophysiological recordings in brain slices we found that a focus of epileptiform discharges developed within 4 -7 d after treatment and could be recorded up to 49 d postoperatively in Ͼ60% of slices from treated animals but only rarely (10%) in sham-operated controls. Epileptiform activity involved both glutamatergic and GABAergic neurotransmission. Epileptiform activity was also induced by direct cortical application of native serum, denatured serum, or albumin-containing solution. In contrast, perfusion with serum-adapted electrolyte solution did not induce abnormal activity, thereby suggesting that the exposure of the serum-devoid brain environment to serum proteins underlies epileptogenesis in the blood-brain barrier-disrupted cortex. Although many neuropathologies entail a compromised bloodbrain barrier, this is the first direct evidence that it may have a role in the pathogenesis of focal cortical epilepsy, a common neurological disease.

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.

The effect of low extracellular Mg2+ concentration ([Mg2+]o) on neuronal activity was studied in rat hippocampal slices. After 20-40 min of perfusion with Mg2+-free medium, when [Mg2+]o declined to approximately 0.1-0.4 mM, spontaneous field potentials developed in the CA1 and CA3 regions, but not in the dentate gyrus. In the CA3 pyramidal cell layer, these potentials consisted of repetitive (0.3-0.5 Hz), 40- to 120-ms-long positive deflections (2-5 mV) with superimposed population spikes. In the stratum (str.) pyramidale of the CA1 region, positive-negative deflections (less than 3 mV) lasting for 30-80 ms were observed, which occurred with a frequency of 0.3-0.5 Hz. In some cases, longer lasting and rapidly recurring events were also observed. In CA3 pyramidal cells, the intracellular correlates of the field potential transients were 20- to 30-mV paroxysmal depolarization shifts (PDS) with superimposed bursts of action potentials, followed by large (greater than 10 mV), 500- to 1,200-ms-long afterhyperpolarizations (AHP). In contrast, pyramidal neurons of the CA1 area did not show PDSs; instead, sequences of excitatory postsynaptic potentials (EPSPs)/inhibitory postsynaptic potentials (IPSPs) accompanied the transient field potential changes. Occasionally, spontaneous EPSPs/IPSPs, occurring with high frequencies, could also be observed in CA1 without any field potential transients. In both hippocampal regions, the epileptiform activity evolved without significant alterations in the resting membrane potential (RMP) and input resistance (RN) of the neurons, although a 2- to 5-mV reduction in action potential threshold was noted. The spontaneous activity in Mg2+-free medium was readily suppressed by raising the extracellular Ca2+ concentration ([Ca2+]o) from 1.6 to 3.6 mM. The perfusion of 10-30 microns DL-2-amino-5-phosphonovaleric acid (2-APV), an antagonist for the glutamate receptors of the N-methyl-D-aspartate (NMDA) type, also attenuated or reversibly blocked the spontaneous activity. Surgical isolation of area CA1 from CA3 ceased the occurrence of the transients in CA1 but not in CA3. The synaptic input/output curves were shifted to the left in the absence of [Mg2+]o. Threshold intensity for eliciting population spikes was 50-75% of that in normal medium. Paired-pulse facilitation was still present near threshold, but was reduced at higher stimulus intensities. Decreases in [Ca2+]o, produced by repetitive stimulation (20-Hz/5-10 s) of the Schaffer collateral/commissural pathway and monitored with ion-selective microelectrodes in the CA1 region, were enhanced in Mg2+-free medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Using a human glial fibrillary acidic protein (hGFAP) promoter-driven cre transgene, we have achieved efficient inactivation of a floxed connexin43 (Cx43) gene in astrocytes of adult mice. The loss of Cx43 expression was monitored in a cell-autonomous manner via conditional replacement of the Cx43-coding region by a lacZ reporter gene. In this way, we bypassed the early postnatal lethality previously reported for Cx43 null mice and characterized the phenotypic consequences of Cx43 deficiency in the CNS. Mice lacking Cx43 in astrocytes were viable and showed no evidence of either neurodegeneration or astrogliosis. Spreading depression (SD) is a pathophysiological phenomenon observed in the CNS that is characterized by a propagating wave of depolarization followed by neuronal inactivation. Inhibitors of gap junctional communication have previously been shown to block initiation and propagation of SD. In contrast, we observed an increase in the velocity of hippocampal SD in the stratum radiatum of mice lacking Cx43 in astrocytes. In the same brain subregion, dye-coupling experiments revealed a reduction in overall astrocytic intercellular communication by approximately 50%. This strongly suggests separate and different neuronal and glial contributions of gap junctional intercellular communication to SD. Concomitant with increased velocity of spreading depression, we observed enhanced locomotory activity in mice lacking Cx43 in astrocytes.