Laura Spilker

Home Institution
Humboldt-Universität Berlin

Host Institution
The Children's Hospital of Philadelphia, Department of Neurology Research Mentor: Douglas A. Coulter, Ph.D.

E-Mail: laura.spilker@gmx.de

Research Topic
see Abstract
Personal Reactions to the U.S. Experience
Philadelphia is a fun city and I felt at home right away, which was in large part due to living at International House, sharing a bathroom and a kitchen with 9 great girls from all around the world. I am fortunate to work in a great lab with very skilled and helpful people. I enjoy a lot of freedom in my scientific work, which is a fantastic experience, even though I find it sometimes very challenging to spent a lot of money and be completely responsible for what I do and how my experiments turn out. I am definitely learning a lot and I am very happy to have the chance to work here.
Greatest Difficulties Encountered
It always takes a lot of patience and good-will to deal with any type of administrative business - but stay calm - losing your temper will only make it worse.
Most humorous incident
1) I was not able to receive an e-mail to my CHOP e-mail address because it contained "inappropriate language" (the bad bad "F -word"). Only after I called the Information Security Department and let someone read it for approval was I able to get hold of it.
2) Going shopping for winter shoes with someone who has never experienced cold, snow or ice before.

Helpful Hints for Future Students
- Try to live in an international student-living home - after all, ethnic diversity and meeting people from everywhere is what the U.S. is about.
- Find out about talks or Grand Rounds - you might get the chance to hear and meet renowned scientists and often free food is provided.
- Check out www.ldpost.com for cheap rates for international calls.

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Abstract on Research Topic
Transcriptional regulation of GABAA receptor expression and functional consequences after kainic acid (KA) treatment in organotypic hippocampal slice culture

Authors
Laura A. Spilker, Tatiana Y. Rikhter and Douglas A. Coulter
Institution
Pediatric Regional Epilepsy Program of the Children's Hospital of Philadelphia and the Joseph Stokes Jr. Research Institute of The Children's Hospital of Philadelphia, Philadelphia, USA
Introduction
Status epilepticus (SE) can lead to long-term changes in protein expression and function in hippocampal neurons and may thereby mediate temporal lobe epilepsy (TLE) - a disorder characterized by cell loss, mossy fiber sprouting, hippocampal and mesial temporal sclerosis and spontaneous recurrent seizures. A region of the hippocampus early affected by activity-induced changes is the dentate gyrus (DG). The DG exerts "gate keeper" function controlling propagation of synaptic activity to hippocampal regions CA1 and CA3. Changes in synaptic behavior of dentate granule cells (DGCs) can promote increased seizure susceptibility. GABAA receptor subunit mRNA expression is altered and total amount is downregulated to 26 % of control levels in DGCs in rats 24 h after induction of SE by injection of pilocarpine (Brooks-Kayal et al. 1998, Rikhter et al. submitted). This leads to altered frequency, kinetics, pharmacology and amplitude of miniature postsynaptic inhibitory currents (mIPSCs). Total GABAA receptor mRNA expression rebounds at 2 weeks after SE. Little is known about the transcriptional regulation of mRNA encoding neurotransmitter receptor (NR) proteins. GABAA receptor genes contain the neuron-restrictive silencing element (NSRE or RE1) a regulatory region within their promoters which binds the corresponding RE1-silencing transcription factor (REST). Interaction of these two compounds activates histone deacetylase (HDAC) leading to condensation of chromatin restricting access of transcription factors. Silencing of genes containing NSRE can be prevented by HDAC inhibitors like Trichostatin and the anticonvulsant Valproic Acid (Phiel et al. 2001). Regulatory effects of SE on the NRSE/REST system have been studied in the hippocampus. KA treatment induces upregulation of REST 4 h post-SE in the dentate gyrus (Palm et al. 1998). Hypo- and hyperacetylation of histones precede downregulation of GluR2 mRNA and upregulation of brain-derievd neurotrophic factor in CA3 neurones as early as 3 h after pilocarpine treatment (Huang et al. 2002). The aim of this study is to investigate if manipulation of HDAC activity by Valproic Acid and Trichostatin can prevent downregulation of GABAA receptor mRNA expression providing a therapeutic strategy to protect neurons from undergoing changes, which lead to manifestation of epilepsy. The culture model offers the possibility to depict a detailed time course of molecular and functional changes.
Methods
Organotypic hippocampal slice cultures are prepared from 10 to 13 days old rat pups (P10 - P13). Hippocampi are dissected and cut into 400-µM slices. Slices are placed on semipermeable membrane inserts and medium is exchanged every other day. Cultures are exposed to 6 µM KA for 4, 24 and 48 hours at 10 days in vitro to induce hyperexcitability. After treatment inserts are transferred to fresh medium which is exchanged at 1 h and 24 h intervals to ensure complete washout of KA (see Routbort et al. 1999). This procedure serves as a model of distinct exposure to elevated synaptic activity as found in SE in in vivo models. Slices are transferred to a submerged recording chamber and perfused with artificial cerebrospinal fluid (aCSF). Whole cell patch clamp recordings are performed at room temperature of visually identified DGCs 1h after onset of KA application, 1 h after and then every other day (from day 1 to 15) after exposure. To isolate mIPSCs the aCSF contains DNQX and AP-5 (10 and 20 µM) to block excitatory synaptic responses and tetrodotoxin (TTX) to inhibit voltage dependent sodium currents. The electrodes are filled with a solution containing Cesium and QX-314 to block GABAB receptor mediated postsynaptic events and RNAsin to prevent digestion of mRNA. To assay changes in transcription cells are harvested and collected in eppendorf tubes after recording. Two rounds of aRNA amplification are performed (Eberwine et al. 1992). The samples are labeled with a-32P-CTP and hybridized with reverse Northern slot blots. Slot blots are prepared containing GABAA receptor clones. All DNAs were sequenced to ensure identity and are linearized by restriction digestion and 1 µg of each is applied to a nylon membrane in a slot-blot apparatus. After hybridization for 40 h at 42º C in a formamide-containing buffer, slot-blots are washed and exposed to a Phosphorimager screen for quantification of RNA. Recordings as well as mRNA expression profiles are performed from day 1 to 14 after KA treatment for close observation of SE-induced alterations.
Progress
So far hippocampal slices have been prepared and were maintained in culture for 3 weeks. Miniature IPCs of DGCs were recorded after 10 days in vitro. Frequency, amplitude and current kinetics (time to peak and decay time) showed values similar to those observed in acute slices and dissociated neurons. Also 7 dissociated neurons were harvested for practice and successfully amplified. The next step will be to validate the KA treatment protocol in cultures.