Interneuron Progenitors Attenuate the Power of Acute Focal Ictal Discharges

Estanislao De la Cruz Mingrui Zhao Lihua Guo Hongtao Ma Stewart A. Anderson Theodore H. Schwartz 0 ) Department of Psychiatry, Weill Cornell Medical College, New York Presbyterian Hospital , New York, NY 10065, USA Interneuron progenitors from the embryonic medial ganglionic eminence (MGE) can migrate, differentiate, and enhance local inhibition after transplantation into the postnatal cortex. Whether grafted MGE cells can reduce ictal activity in adult neocortex is unknown. We transplanted live MGE or killed cells (control) from pan green fluorescent protein expressing mice into adult mouse sensorimotor cortex. One week, 2 and 1/2 weeks, or 6 to 8 weeks after transplant, acute focal ictal epileptiform discharges were induced by injection of 4-aminopyridine (4-AP) 2 mm away from the site of transplantation. The local field potential of the events was recorded with 2 electrodes, 1 located in the 4-AP focus and the other 1 in the transplantation site. In all control groups and in the 1-week live cell transplant, 4-AP ictal discharges revealed no attenuation in power and duration from the onset site to the site of transplantation. However, 2.5 or 6~8 weeks after MGE Estanislao De la Cruz and Mingrui Zhao contributed equally to this study transplants, there was a dramatic decrease in local field potential power at the MGE transplanted site with little decrease in ictal duration. Surprisingly, there was no relationship between grafted cell distribution or density and the degree of attenuation. As remarkably low graft densities still significantly reduced discharge power, these data provide further support for the therapeutic potential of interneuron precursor transplants in the treatment of neocortical epilepsy. - It is estimated that more than 30% of all epilepsy patients are refractory to conventional anti-epileptic medication [1]. A proportion of these patients may be candidates for surgery to remove the epileptic focus [1, 2], but a significant fraction continue to have seizures without hope of further treatment. The loss of neuronal inhibition has been demonstrated in a variety of epilepsy models and proposed as a possible mechanism for increased excitability. In fact, several interneuron subgroups have been associated with the suppression of seizures, including chandelier cells expressing parvalbumin (PV) [3] and basket cells expressing either somatostatin (SST), and/or neuropeptide Y [4]. As a result, transplantation of inhibitory interneurons into an epileptic focus may be a potential therapy for cases of pharmacoresistant epilepsy [5]. A variety of transplantation strategies have been used to suppress epileptic activity, such as the use of immortal cell lines engineered to produce gamma-Aminobutyric acid (GABA) [6], and embryonic stem cell-derived adipose tissue engineered to release the potent anti-epileptic adenosine [7, 8]. However, only recently have advances in understanding cortical interneuron origins [9] and appreciation of their remarkable ability to migrate and survive after transplantation into neonatal or adult cortex [10] ultimately led to interneuron transplantation studies to treat seizures in rodents [1113]. Although species differences with primates appear to exist, in rodents and ferrets most cortical interneurons, including the PV and the SST expressing subgroups, originate in the medial ganglionic eminence (MGE) of the subcortical telencephalon [14]. Genetic fatemapping and transplantation studies indicate that within the MGE there is a bias for PV or SST expressing interneurons to originate from ventral or dorsal MGE regions, respectively [1517]. Transplanted MGE cells not only differentiate into mature GABAergic (gamma-aminobutyric-acid-releasing) interneurons, but also form inhibitory synapses that increase GABAergic synaptic transmission onto adjacent pyramidal cells [18] and alter cortical pyramidal neuron plasticity [19]. Three recent studies have indeed presented evidence that transplantation of MGE-derived interneuron precursors can reduce seizure activity. In the first study, transplants into neonatal neocortex reduced generalized seizures and modestly improved survival in a genetic model of cortical hyperexcitability [12]. In the second study, reduced seizure threshold produced in adult mice by selective killing of hippocampal interneurons was normalized by transplantation of MGE-derived interneurons [13]. Finally, MGEderived neural stem cells transplanted into adult hippocampus reduced kainic acid-induced kindled seizures, although in this study the grafts included many astrocytes [11]. Despite these advances, important questions remain about this promising therapy. For example, how soon after transplantation do the anti-seizure effects appear? Is this therapy use effective against other types of ictal events, such as focal neocortical seizures? Finally, is there a rapid method to screen transplant efficacy in an acute in vivo model? To address these questions, we investigated the relationship between the quantity of surviving MGE transplants and their anti-epileptic effect using a model of focal injection of 4-aminopyridine (4-AP), which is a potent convulsant that acts by blocking slowly inactivating potassium currents [20] and enhancing the release of synaptic neurotransmitters [21], which elicits focal ictallike events that initiate spontaneously and propagate horizontally [22]. Although the pharmacoresistance of focal neocortical 4-AP injection in vivo has not been tested, intraperitoneal injection and in vitro data indicate at least partial pharmacoresistance [2327]. Although the 4-AP model is not a model of chronic epilepsy, it is advantageous as a rapid throughput screen of anti-epilectic therapy [28]. Herein, we describe that MGE transplants dramatically reduce seizure propagation indicating that acute focal injection of 4-AP can be used as a high throughput model to assess cell transplant therapeutic efficacy. Materials and Methods In Vivo Transplantation All experimental procedures were approved by the Weill Cornell Medical College Animal Care and Use Committee following the National Institutes of Health guidelines. Green fluorescent protein (GFP) expressing transgenic mice were maintained on a CD1 background. At 13 days of gestation (E13.5), dams were sacrificed and the GFP + embryos were placed in ice-cold Hank's buffer. The brains were removed and the ventral forebrain was exposed by removing the dorsal and lateral region of cortex. MGE cells are then harvested from 250-m sections by first gently freeing the dorsal and ventral MGE from the adjacent tissue and then by mechanically dissociation (Fig. 1). Cells were then re-suspended in Neurobasal/B27 medium (Gibco/Invitrogen Grand Island, NY) and kept on ice until transplantation. Adult CD1 mice 6 to 8 weeks old were initially anesthetized with 1.5 to 2% isoflurane in 70% N2:30% O2 by face mask and mounted in a stereotaxic frame. Mice were maintained on 1.5% isof (...truncated)