Modulation of Calcium-Dependent Inactivation Figure 4. b2AR specifically modulates CDI in TC neurons of dLGN. (A) Representative current traces recorded under control conditions (upper left panel) and during extracellular application of specific b2AR agonist salmeterol (10 mM) plus antagonists for the other two types of receptor (CGP 20712 dihydrochloride-b1 antagonist and SR 59230A hydrochloride-b3-antagonist, 100 mM each; lower left panel) elicited by the indicated pulse protocol (middle left panel). The bar graph (right panel) represents Dinact under different recording conditions (as indicated). The mean value of four cells recorded under control conditions was taken for comparison with four cells recorded under 10 mM salmeterol plus 100 mM b1+b3 antagonists. Data are presented as means 6 SEM of several independent experiments. **P,0.01. Significance of salmeterol plus antagonists versus control was calculated by Student’s t test. The degree of inactivation is given by the normalized current amplitude of the mean postpulse I/V at +10 mV. (B) Immunocytochemical analysis of primary cultures of the dorsal thalamus using MAP2 specific antibody (left upper panel, red) and b2AR specific antibody (left down panel, green). The merged picture revealed the co-expression of these two proteins in somatic regions and proximal dendrites. Enlarged inlay represents magnification of the area indicated by the rectangle. Yellow dots represent places were these two proteins are in close proximity. Data shown are representative pictures from several independent immunostainings and preparations of neurons. In all cases, omission of primary antibodies resulted in no fluorescence signal above background (negative control). doi:10.1371/journal.pone.0027474.g004 modulation of CDI in TC neurons after bAR stimulation. The double-pulse protocol was applied using the phosphatase inhibitor okadaic acid (OA; 10 mM alone; p.0.05; Figure 8A), or in combination with isoproterenol (10 mM; Figure 8B). After blocking dephosphorylation processes, the degree of inactivation is reduced to 29.361.1% (n = 5) in comparison to bAR stimulation alone (35.762%, n = 5; p ,0.01), and non-stimulated control cells which showed Dinact of 4063.3% (n = 5; p ,0.001) in these experiments. Next, the expression of protein phosphatases PP2A was investigated in 10 days old cultured thalamic neurons by using antibodies for PP2A (Figure 8C, upper right panel) and CaV1.2 PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e27474