Larry S. Benardo

GABAergic Cell Grafts Control Seizure Activity

PURPOSE: Cell transplantation into the brain is an aggressive clinical alternative. The hopes of treating diseases like intractable temporal lobe epilepsy have been subdued because the preclinical successes thus far have shown only slowing of epileptogenesis, or suppression of electrically induced seizures. Because the hallmark of epilepsy is spontaneous seizures, the clinical relevance of these studies has been questioned. The purpose of this study was to establish that cells genetically engineered to produce γ -aminobutyric acid (GABA) could suppress spontaneous seizures in an accepted model of temporal lobe epilepsy. METHODS: Conditionally immortalized neurons were engineered to produce GABA under the control of tetracycline. These cells were transplanted into the substantia nigra of spontaneously seizing animals. After transplantation, the animals were monitored for 3 days immediately after surgery and again for 3 days beginning 7 to 8 days after surgery. Seizures and epileptiform spikes were recorded and later analyzed with detection software combined with video monitoring. RESULTS: Animals that received genetically engineered GABA-producing cells had significantly fewer spontaneous seizures than did animals that received control cells, or animals that received GABA-producing cells plus doxycycline at the observation period starting 1 week after transplantation. A significant suppression of epileptiform spikes also was noted between the group that received GABA-producing cells and the group that received the same cells but were given doxycycline. The engineered cells show evidence of integration with the host but limited survival. CONCLUSIONS: These data demonstrate that genetically engineered cells have the ability to suppress spontaneous seizures when transplanted into seizuremodulating nuclei. This is an important step toward defining a clinical potential for this approach in epilepsy. The fact that the gene of interest can be regulated suggests that individualizing transplant therapy may be possible.

Altered Sodium Channels Underlie Anticonvulsant Drug Insensitivity.

The development of resistance to pharmacologic treatment is common to many human diseases. In chronic epilepsy, many patients develop resistance to anticonvulsant drug treatment during the course of their disease, with the underlying mechanisms remaining unclear. We have studied cellular mechanisms underlying drug resistance in resected hippocampal tissue from patients with temporal lobe epilepsy by comparing two groups of patients, the first displaying a clinical response to the anticonvulsant carbamazepine (CBZ) and a second group with therapy-resistant seizures. With patchclamp recordings, we show that the mechanism of action of CBZ, use-dependent block of voltage-dependent Na+ channels, is completely lost in CBZ-resistant patients. Likewise, seizure activity elicited in human hippocampal slices is insensitive to CBZ. In marked contrast, CBZ-induced use-dependent block of Na+ channels and blocked seizure activity in vitro in patients clinically responsive to this drug. Consistent with these results in human patients, we also show that use-dependent block of Na+ channels by CBZ is absent in chronic experimental epilepsy. Taken together, these data suggest that a loss of Na+-channel drug sensitivity may constitute a novel mechanism underlying the development of drugresistant epilepsy.

Synchronized GABAergic Inhibition Drives Epileptiform Activity

We studied cellular mechanisms of synchronization of epileptiform activity induced by kainic acid in a novel preparation of superfused rat hippocampus in vivo. Under urethane anesthesia, kainate induced epileptic population spikes occurring at 30 to 40 Hz. Pyramidal cells fired exclusively during population spikes, with an average probability of 0.34 on rebound of rhythmic γ aminobutyric acid (GABA)A-mediated inhibitory postsynaptic events. Excitatory synaptic events contributed little to seizure activity. Rhythmic epileptiform activity was suppressed by blocking GABAA receptors and was slowed by barbiturates. Thus GABAergic inhibition is instrumental in synchronizing kainate-induced epileptiform rhythmic activity in the γ frequency band in the intact hippocampus in vivo.

Gap Junctions in Epileptogenesis: Chicken or Egg?

Prolonged (18-hour) exposure of cultured hippocampal slices to the type-A γ -aminobutyric acid (GABA)receptor blocker, bicuculline methiodide (BMI), 10 μm, increased the levels of connexin 43 (Cx43) and connexin 32 (Cx32) mRNAs, but not connexin 26 and connexin 36, as demonstrated by RNase protection assays. The levels of Cx43 and Cx32 proteins in membrane fractions detected by Western blotting also were significantly increased. Immunoblotting indicated that BMI also promoted a significant expression of the transcription protein c-fos. The rate of fluorescence recovery after photobleaching, an index of gap-junctional coupling, also was significantly increased, whereas it was blocked by the gap-junctional blocker, carbenoxolone (100 μm). Extracellular recordings in CA1 stratum pyramidale, performed in BMI-free solution, demonstrated that BMIexposed cultures possessed synaptic responses characteristic of epileptiform discharges: (a) significantly greater frequency of spontaneous epileptiform discharges, (b) postsynaptic potentials with multiple population spikes, and (c) significantly longer duration of primary afterdischarges. Carbenoxolone (100 μm), but not its inactive analogue, oleanolic acid (100 μm), reversibly inhibited spontaneous and evoked epileptiform discharges. The findings of BMI-induced parallel increases in levels of gap-junction expression and function, and the increase in epileptiform discharges, which were sensitive to gap-junctional blockers, are consistent with the hypothesis that increased gap-junctional communication plays an intrinsic role in the epileptogenic process. COMMENTARY

Adenosine Kinase Elevation Increases Activity in the Kainic Acid Model of Epilepsy

Endogenous adenosine in the brain is thought to prevent the development and spread of seizures via a tonic anticonvulsant effect. Brain levels of adenosine are regulated primarily by the activity of adenosine kinase. To establish a link between adenosine kinase expression and seizure activity, we analyzed the expression of adenosine kinase in the brain of control mice and in a kainic acid– induced mouse model of mesial temporal lobe epilepsy. Immunohistochemical analysis of brain sections of control mice revealed intense staining for adenosine kinase, mainly in astrocytes, which were more or less evenly distributed throughout the brain, as well as in some neurons, particularly in olfactory bulb, striatum, and brainstem. In contrast, hippocampi lesioned by a unilateral kainic acid injection displayed profound astrogliosis and therefore a significant increase in adenosine kinase immunoreactivity accompanied by a corresponding increase of enzyme activity, which paralleled chronic recurrent seizure activity in this brain region. Accordingly, seizures and interictal spikes were suppressed by the injection of a low dose of the adenosine kinase inhibitor 5-iodotubercidin. We conclude that overexpression of adenosine kinase in discrete parts of the epileptic hippocampus may contribute to the development and progression of seizure activity.

Metabotropic Glutamate Receptors and Epileptiform Bursting.

Differential Roles for mGluR1 and mGluR5 in the Persistent Prolongation of Epileptiform BurstsMerlin LRJ Neurophysiol 2002;87:621–625PurposeTransient activation of group I metabotropic glutamate receptors (mGluRs) with the selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) produces persistent prolongation of epileptiform bursts in guinea-pig hippocampal slices, the maintenance of which can be reversibly suppressed with group I mGluR antagonists. To determine the relative roles of mGluR1 and mGluR5 in these group I mGluR-dependent induction and maintenance processes, subtype-selective antagonists were used. In the presence of picrotoxin, DHPG (50 μM, 20–45 min) converted interictal bursts into 1- to 3-s discharges that persisted for hours after washout of the mGluR agonist. 2-Methyl-6–(phenylethynyl)-pyridine (MPEP, an mGluR5 antagonist; 25 μM) and (+)-2-methyl-4-carboxy-phenylglycine (LY367385, an mGluR1 antagonist; 20–25 (μM) each significantly suppressed the ongoing expression of the mGluR-induced ...

GABAergic Interneuron Reorganization During the Late Period May Contribute to Hippocampal Epileptogenesis

Alterations of Hippocampal GABAergic System Contribute to Development of Spontaneous Recurrent Seizures in the Rat Lithium-Pilocarpine Model of Temporal Lobe EpilepsysAndre V, Marescaux C, Nehlig A, Fritschy JMHippocampus 2001;11:452–468Reorganization of excitatory and inhibitory circuits in the hippocampal formation following seizure-induced neuronal loss has been proposed to underlie the development of chronic seizures in temporal lobe epilepsy (TLE). Here, we investigated whether specific morphological alterations of the GABAergic system can be related to the onset of spontaneous recurrent seizures (SRS) in the rat lithium-pilocarpine model of TLE. Immunohistochemical staining for markers of interneurons and their projections, including parvalbumin (PV), calretinin (CR), calbindin (CB), glutamic acid decarboxylase (GAD), and type 1 GABA transporter (GAT1), was performed in brain sections of rats treated with lithium-pilocarpine and sacrificed after 24 h, during the silent phase (6 and 12 days), or afte...