Modulation of Calcium-Dependent Inactivation Figure 2. Single cell and qRT-PCR expression profiles of AKAP5, AKAP7 and the Gs subunit of G proteins. (A) Two different cell types are expressed in LGN, interneurons that are GAD67 positive and TC neurons that are GAD67 negative. These types of cells were collected under visual control and classified based on their morphological differences. Thereafter, mRNA was extracted. Representative PCR gel photo showing bands specific for AKAP5, AKAP7, and the Gs subunit of G proteins. Note that AKAP5 is only expressed in TC neurons while AKAP7 was not detectable. (B) mRNAs were isolated from the indicated brain tissues. qRT-PCR analysis showed expression of both AKAP5 and AKAP7 genes in the dLGN. doi:10.1371/journal.pone.0027474.g002 Preparation and culturing of dissociated cell cultures from the dorsal thalamus and hippocampus Dorsal thalami were prepared from embryos (Long-Evans rats) at stage E19 and subsequently transferred into ice-cold HBSS without Ca2+/Mg2+. After triple washing with HBSS (5 ml), 2.0 ml HBSS containing 0.5% trypsin was added to the tissue, followed by incubation at 37uC for 20 min. Thereafter tissue was washed again five times with 5 ml HBSS and finally transferred into tubes (2 ml) with HBSS containing 0.01% DNaseI. For dissociation thalamic tissue was pressed slowly three times through a 0.9 mm gauge needle followed by three passages through a 0.45 mm gauge needle. The remaining cell suspension was poured through a nylon tissue into a 50 ml tube and filled up with 18 ml DMEM. After estimating cell quantity, the suspension was diluted with DMEM in order to achieve a cell density of 30,000 cells/ml. A 500 ml aliquot of this suspension was placed on each well of a Figure 3. Identification of CDI in TC neurons. (A) Scheme of the double-pulse protocol used to elicit HVA Ca2+ currents. TC neurons were held at 240 mV and conditioning pulses to varying potentials (240 to +60 mV, 200 ms duration) were followed by a brief gap (240 mV, 50 ms) and a subsequent analyzing test pulse to a fixed potential of +10 mV (200 ms). (B) Family of representative current traces elicited by the pulse protocol shown in (A). (C) Dinact equation for calculating effects of CDI in TC neurons. doi:10.1371/journal.pone.0027474.g003 PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e27474