200 mA). The transfer buffer contained 25 mM Tris, 192 mM glycine, 0.02% SDS and 20% methanol. After blotting, mem- branes were blocked with 5% dry milk and 0.1% Tween 20 in TBS for 2 h and subsequently incubated at 4uC overnight with a specific dilution of antibodies in TBSA containing 0.1% Tween 20. After final washing steps, the blots were incubated with HRP- conjugated secondary antibodies (1:5000) for 2 h, washed again and finally developed using ECL films. Microscopy Immunofluorescence analysis of cultured neurons was done using a computer-controlled inverted laser scanning microscope (Leica, Bensheim, Germany) allowing recording of Z-stacks and enabling 3D deconvolution of the obtained images. Image analysis was done using MetaMorph, ImageJ (NIH), and Adobe Photoshop CS (version 9.0 CS2) software. Data analysis Statistical data analysis was done by Student’s t test or one way ANOVA as indicated using GraphPad Prism 5 and Microcal Origin 6. All values were presented as mean 6 SEM. As we were able to demonstrate a Gaussian distribution for the three main parameters (current amplitude, Dinact and ratio of inactivation) analyzed in the present study under control conditions, statistical significance was evaluated by Student’s t-test. Where applicable, control values were compared with corresponding values obtained during drug application for the same cells. When intracellular substance application had to be used, an appropriate number of control cells was recorded from the same slice and used for statistical comparison. Values of P,0.05 were considered statistically significant. Results PCR expression patterns of the main components of the bAR signaling cascade in dLGN In a first attempt to investigate the modulation of CDI via bARs, we analyzed the expression patterns of the main components of the proposed b-AR signaling pathway in dLGN TC neurons by performing RT-PCR analyses on a tissue and single cell level. Expression of RNA of the main neuronal L-type Ca2+ channels (CaV1.2/a1C, CaV1.3/a1D) were found in whole brain samples (Figure 1A), the VB (Figure 1B), and dLGN (Figure 1F). The skeletal muscle type CaV1.1/a1S and the retina- specific CaV1.4/a1F were not detected in thalamic tissue (Figure 1), indicating a specific RT-PCR amplification. Further- more, the specific expression of main components of the bAR signaling cascade supposed to be involved in CDI modulation in TC neurons was confirmed in dLGN tissue (Figure 1C–1E). The expression of AKAP and the stimulatory G protein (Gs) was probed in a cell type-specific manner using single identified cells obtained from dLGN tissue. Acutely dissociated cells were observed under an inverted microscope and small bipolar interneurons and larger multipolar TC neurons were visually identified by using established criteria [22,23]. Single cells were collected by means of a suction pipette and used for standard PCR or quantitative real-time PCR (qRT-PCR). Because of very low amounts of the mRNA species targeted here, in some sets of experiments it was necessary to pool up to 10 cells. As Figure 2A shows, in three independent experiments (n = 3) the Gs subunit was expressed in both types of neurons, AKAP5 was only expressed in TC neurons, and AKAP7 was not detected at the single cell level. To obtain larger amounts of mRNA, we also performed qRT-PCR with samples from dLGN tissue. Using Modulation of Calcium-Dependent Inactivation specific primers for AKAP5 and AKAP7, we were able to detect expression signals for these two genes in dLGN with almost identical expression levels (Figure 2B). Of note, that while AKAP7 is nearly equally expressed in the brain, AKAP5 exhibited a slightly higher expression level in the primary somatosensory cortex and hippocampus (data not shown). In view of positive signals for both AKAP subtypes from dLGN tissue and AKAP5 in single TC neurons, the absence of positive PCR bands for AKAP7 in extracts from single cells indicted that the amount of mRNA was below the detection limit of the method used here or pointed to an expression in other cell types present in intact tissue (glia cells, endothelial cells, blood cells). CDI is active in TC neurons in brain slices Next, we demonstrated the occurrence of CDI in TC neurons in brain slices. Total HVA Ca2+ current, which is composed of about 40% current through L-type channels, was measured in the presence of TTX (1 mM) [24]. We have shown before that blocking of L-type calcium channels using nifedipine (1 mM), (n = 8) indeed significantly reduced CDI [5]. HVA Ca2+ currents were recorded in 160 cells and a double pulse voltage protocol (Figure 3A) was used to effectively disclose CDI [3]. If CDI is operative, the current evoked by the test pulse should exhibit a U- shaped dependence on the conditioning pulse potential, with maximal inactivation occurring at the peak of the conditioning pulse current-voltage (I–V) relationship. Under standard condi- tions the I–V relationship of the conditioning pulse demonstrated HVA Ca2+ currents with an activation threshold of –40 mV, maximal inward current at around +10 mV, and an apparent reversal potential at around +60 mV (data not shown). The test pulse I–V (peak amplitude of the test pulse current plotted vs. conditioning pulse voltage) revealed minimal current occurring at +10 mV (Figure 3C) as expected for a CDI mechanism. With respect to the amplitude of the test pulse current elicited from the holding potential of –40 mV, the degree of inactivation (Dinact) was 39.5 6 0.5% (n = 100; Figure 3C). By default, the test pulse I–V was obtained with the conditioning pulses altered in 10 mV increments from 240 mV (Figure 3A). I–V relationships were obtained as described before and all experiments were done using a protocol resulting in maximal CDI [5]. Taken together, these findings demonstrate a CDI mechanism in TC neurons in the slice preparation that is very similar to that described in these cells after acute isolation [9]. The role of b-AR in CDI modulation of L-type Ca2+ channels Since activation of the cAMP/PKA pathway is able to reduce CDI in isolated TC neurons [9,10], activation of bARs is expected to modulate CDI of L-type calcium channels in the dLGN. bARs consist of three similar receptor subtypes (b1, b , b3). Using 2 commercially available pharmacological substances, we specifical- ly stimulated and blocked receptor subtypes and evaluated their possible relevance for CDI modulation. First, we used general agonists and antagonists for bARs, namely isoproterenol and propranolol (data not shown). Experiments testing bAR activation alone using 10 mM isoproterenol revealed an inhibiting influence of bAR stimulation on CDI. Dinact was decreased to 35.762% (n = 4, p.0.05) as compared to control (Dinact = 40.163%). The effect of isoproterenol on CDI was reversed by co-application of the bAR antagonist propranolol (100 mM, Dinact = 43.762.1, n = 5; p.0.05; control Dinact = 46.161.6%, n = 5; data not shown). These experiments demonstrate a general role of bAR in modulation of CDI in TC neurons. The rather moderate effect PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e27474