Each slice culture was visually inspected using IR-DIC (Fig
Each slice culture was visually inspected using IR-DIC (Fig. that interictal-like spikes precede the appearance of ictal-like activity in a reduced in vitro preparation. Epileptiform activity in cultures resembledin vivoepilepsy, including sensitivity to anticonvulsants and steadily increasing seizure incidence over time, although seizure frequency and rate of epileptogenesis were higherin vitro. Organotypic hippocampal slice cultures comprise a useful model system for investigating mechanisms of epileptogenesis as well as developing anti-epileptic and anti-epileptogenic drugs. Keywords:Epileptogenesis, growth medium, interictal spikes, ictal discharges, organotypic hippocampal slices, phenytoin == Introduction == Although electrographic interictal spikes are Nicarbazin so reliably associated with a propensity for spontaneous seizures that their presence is used diagnostically (Engel, 1989), the reasons for this robust association are entirely unknown (de Curtis and Avanzini, 2001). We recently hypothesized that interictal spikes might drive epileptic circuit formation (Staley et al., 2005). One characteristic of epileptogenesis is the appearance of axons returning to excite neurons in their network of origin, known as sprouting(Sutula and Dudek, 2007). Spikes are the synchronous discharge of neuron populations, an activity possibly guiding the synapse formation of newly sprouted axons within their circuit of origin following the developmental fire together, wire together axiom. Once formed, spike timing-dependent plasticity rules would predict that such synapses would be strengthened by Nicarbazin synchronous activation of the population during spiking, a mechanism observed in a variety Nicarbazin of in vitro preparations (Schneiderman et al., 1994;Bains et al., 1999;Abegg et al., 2004;Behrens et al., 2005;Hellier et al., 2007). The hippocampal organotypic slice culture method (Gahwiler, 1981;Stoppini et al., 1991) is well established and has been used in a wide range of studies, particularly investigations of plasticity of mono-synaptically connected pairs of neurons and pharmacologically induced epileptiform discharges (Gahwiler et al., 1997). While most properties of organotypic hippocampal slice cultures resemble theirin vivocounterparts (Gahwiler, 1984;De Simoni et al., 2003), neurons in this preparation exhibit robust sprouting as a consequence of massive deafferentation and de-efferentation occurring during tissue slicing (Zimmer and Gahwiler, 1984;Frotscher and Gahwiler, 1988). Despite this robust sprouting, however, spontaneous epileptiform activity has rarely been observed (McBain et al., 1989;Lahtinen et al., 2001;Abegg et al., 2004). If spiking drives Rabbit polyclonal to WAS.The Wiskott-Aldrich syndrome (WAS) is a disorder that results from a monogenic defect that hasbeen mapped to the short arm of the X chromosome. WAS is characterized by thrombocytopenia,eczema, defects in cell-mediated and humoral immunity and a propensity for lymphoproliferativedisease. The gene that is mutated in the syndrome encodes a proline-rich protein of unknownfunction designated WAS protein (WASP). A clue to WASP function came from the observationthat T cells from affected males had an irregular cellular morphology and a disarrayed cytoskeletonsuggesting the involvement of WASP in cytoskeletal organization. Close examination of the WASPsequence revealed a putative Cdc42/Rac interacting domain, homologous with those found inPAK65 and ACK. Subsequent investigation has shown WASP to be a true downstream effector ofCdc42 some aspects of epileptogenesis, organotypic slices that developed spiking would be expected to progressively develop ictal-like epileptiform activity, while organotypic slices not developing spikes should not develop any epileptiform activity. We tested this idea using prolonged recordings of population activity from area CA3 under perfusion with complete growth medium at physiological temperature in slice cultures from 730 daysin vitroin the absence of pharmacological manipulations. Our recordings demonstrate an unexpectedly high incidence of spontaneous epileptiform activity. Interictal-like spikes and short bursts of population activity developed before the appearance of ictal-like discharges of more than 3 minutes duration. == Materials and Methods == == Organotypic hippocampal slice cultures roller tube method == Roller-tube type organotypic slice cultures were prepared as described byGhwiler (1981): Briefly, isolated hippocampi from P8 Sprague-Dawley (Charles River Laboratories, Wilmington, MA) rat pups were cut into 350 m slices on a McIlwain tissue chopper (Mickle Lab Eng. Co., Surrey, UK). Slices were mounted in clots of Nicarbazin chicken plasma (Cocalico Biologicals, Reamstown, PA) and thrombin (Sigma-Aldrich, St. Louis, MO) on poly-L-lysine (Sigma-Aldrich, St. Louis, MO) coated glass coverslips (Electron Microscopy Sciences, Hatfield, PA) and incubated in roller tubes (Nunc, Roskilde, Denmark) at 36.