Rhythmic low and high frequency activity in thalamocortical networks depend critically on activation of low- and high-voltage-activated (LVA, HVA) Ca2+ currents. In order to test whether Ca2+ currents are modified during repetitive activation, acutely isolated thalamocortical relay neurons of rats, at postnatal days 12 (P12) to P20, were investigated using patch-clamp, Ca2+ imaging and Western blot techniques. High-voltage-activated, but not LVA Ca2+ currents were reduced significantly during 2 Hz stimulation. Ca2+ imaging experiments demonstrated a close correlation between the increase in intracellular Ca2+ levels and the decrease in HVA Ca2+ current amplitudes. Further examination of HVA Ca2+ currents revealed a 'U-shaped' inactivation curve and a time-dependent inactivation process that could be described by a two-exponential function. The 'U-shape' was significantly reduced, current amplitude was increased significantly and time-dependent inactivation revealed a one-exponential decline with Ba2+ as the charge carrier, following activation of the cAMP/PKA pathway, and following application of phosphatase inhibitors (ascomycin, calyculin A). Western blot analysis and the effect of ascomycin indicated an involvement of calcineurin in the inactivation process. Isolation of HVA Ca2+ current components by subtype-specific blockers revealed that changes in time-dependent inactivation, inactivation curve and current amplitude were carried mainly by L-type and N-type Ca2+ currents. Furthermore, Ca2+-dependent inactivation was operative during stimulation protocols mimicking tonic action potential firing. These data indicate a modulation of L- and N-type Ca2+ channels by phosphorylation, resulting jointly in an increased intracellular Ca2+ influx during activity of the ascending brainstem system, the latter occurring during states of wakefulness.