on CaV1.2 for the scaffold proteins, like AKAP and PSD proteins did not change membrane expression of CaV1.2 [52]. The above findings indicate that binding of these proteins is necessary for the regulation of CaV1.2 after bAR stimulation but does not have an influence on the membrane localization of CaV1.2. Based on the previous findings discussed above and the results presented here, we propose the following role for AKAP in the regulation of CDI in TC neurons. Following bAR stimulation, AC gets activated via G-proteins and produces cAMP which then activates PKA. With support of AKAPs, PKA targets its final effector, the CaV1.2 channels and phosphorylates them (see Figure S1). In the present study we demonstrated that blocking the binding of AKAP to PKA significantly reduces CDI. Moreover, under similar conditions we have shown that PKA phosphoryla- tion of CaV1.2 and translocation of this enzyme close to the channel depends on binding of PKA and AKAPs in hippocampal neurons (see Text S1 & Figure S3). Most recent studies de- monstrated that AKAP5 is required to localize both RIIa and RIIb containing holoenzymes to the dendritic regions of neurons in the hippocampus and striatum. PKA is dramatically delocalized within dendrites in both the KO and D36 (mutant that lacks the PKA binding domain of AKAP5) mice indicating that no other AKAP subtype is able to compensate and maintain normal PKA localization [53]. Our study confirmed an interaction between AKAP and PKA in pull down assays and demonstrated that after stimulation of the bAR signaling cascade a ternary complex is formed between PKA, AKAP, and CaV1.2 and that the formation of this complex is important for CDI modulation in TC neurons. Moreover, as mentioned before, AKAP5 also targets calcineurin which is able to activate protein phosphatase PP1 [54] and therefore might have multiple function in regulation of CDI by influencing both phosphorylation by PKA and dephosphorylation processes. Functional significance of Ca2+ channel phosphorylation after b-AR stimulation Release of transmitters from a number of brainstem terminals modulates the behavioural states of an individual by depolarizing TC relay neurons [37]. During states of slow-wave sleep, thalamic relay neurons are hyperpolarized and display rhythmic burst activity [11]. During states of wakefulness, these cells are de- polarized and display tonic single spike activity, resulting in the faithful transmission of sensory signals through the dorsal thalamus. The shift from burst activity to tonic activity is mediated by increased activity of ascending brainstem fibres that are thought to increasingly release acetylcholine (ACh), noradrenaline (NA) and serotonin (5-HT) during wakefulness. Both via b-receptors and 5-HT via an unknown 5-HT receptor subtype, activate adenylate cyclase [37] in TC relay neurons and are thus able to positively modulate HVA Ca2+ currents. When attempting to integrate the findings of the present study into the known framework of thalamic physiology it can be assumed that HVA Ca2+ currents are especially activated during tonic firing. Furthermore, following release of NA, HVA Ca2+ current amplitudes will be increased while CDI is decreased. Another consequence of bAR stimulation may be the AC/cAMP- dependent inhibition of high conductance Ca2+-dependent K+ (BKCa) channels in sensory TC neurons [55]. In addition, tonic sequences of action potentials are coupled to CICR from intracellular stores, thereby further increasing Ca2+ entry into TC neurons during wakefulness [6]. It has been shown that intracellular Ca2+ release provides Ca2+ that contributes to CDI and activates BKCa channels in TC neurons [5,6]. Computer modelling indicated that activation of BKCa channels leads to the Modulation of Calcium-Dependent Inactivation occurrence of spike frequency adaptation (P. Meuth & T. Budde, unpublished observations), a condition that would impair the faithful 1:1 relay of incoming trains of sensory action potentials. These data indicate a fine-tuned interplay between activity dependent Ca2+ influx, phosphorylation/dephosphorylation pro- cesses and the mode of activity, possibly to enable faithful signal integration and transfer during wakefulness. Future studies will have to unravel the different modulatory pathways that act upstream of the multiple CDI mechanisms thereby pointing to additional functions of CDI and unraveling further the elusive role of HVA Ca2+ channels in thalamic physiology. Supporting Information Text S1 AKAPs assist in PKA translocation from somatic regions to the plasma membrane. Translocation of PKARIIb after bAR stimulation with isoproterenol in hippocampal neurons. (DOC) Figure S1 Main components of the bAR signaling cascade in dLGN. (TIF) Figure S2 Differential expression of CaV2.1 from CaV1.2 in TC neurons of thalamus. Immunocytochemical analysis of primary cultures of the dorsal thalamus using CaV2.1- (green) and MAP2-specific (neuronal marker, red) antibodies. Merge image showed expression of these two proteins in somatic, proximal and in distal regions of TC neurons. Data shown are representative pictures from several independent experiments. In all cases, omission of primary antibodies resulted in staining without signal (negative control). (TIF) Figure S3 PKA translocation from somatic regions to the plasma membrane. (A) Cultured hippocampal cells were treated with 50 mM isoproterenol or kept under control conditions (control/no treatment), fixed and immunostained with PKARIIb antibody. Enlarged inlays represent magnification of the area indicated by the rectangle. Note translocation of PKA from somatic perinuclear region in control cells to more distal regions close to the plasma membrane and probably Ca2+ channels in cells treated with isoproterenol. (B) Ten days old hippocampal cultures were treated for 5 minutes with 50 mM isoproterenol in combination with 50 mM control peptide AKAP St-Ht31 (left panel), 50 mM isoproterenol in combination with 50 mM AKAP St-Ht31 inhibitory peptide (right panel) and 50 mM isoproterenol in combination with 7.5 mM Anisomycine (down panel). The cells were then fixed and immunostained with PKARIIb antibody. Note that PKA is still translocated to proximal dendrites, after blocking the association of PKA with AKAPs, translocation is almost completely inhibited and anisomycine treatment did not block PKA translocation. (C) Quantification of translocation experiments using MetaMorph was done by measurement of PKA distance from centre of the soma to dendrites in pixels after different treatments. Data are presented as means 6 SEM of several independent experiments. ***P,0.001, Anova test. (TIF) Acknowledgments We thank Prof. Volkmar Lessmann for encouragement and support. Thanks are due to A. Jahn, M. Marunde, C. Borutzky and R. Ziegler for excellent technical assistanceand Prof. Oliver Stork (Institute of Biology, Otto-von-Guericke-University, Magdeburg) for his kind help in the microscopy analysis. This work was performed in partial fulfilment of the PhD thesis of V.R. PLoS ONE | www.plosone.org 13 December 2011 | Volume 6 | Issue 12 | e27474