Figure 5. Calindol activation of CaSR expressed in HEK cells occurred with a greater latency than Ca2+ activation of CaSR. A, application of Ca2+ (5 mM) and Calindol (10 mM) at time zero to CaSR- expressing HEK cells in 1 mM Ca2+ and 0 Mg2+ caused a transient increase in fluorescence (F) relative to basal level (F0) indicating an increase in [Ca2+]i. The black curves denote signal from 12 cells and the red curves indicate the average. There was a 5 minute delay between applications. B, average curves from A have been redrawn on the same time-expanded axis to compare effect latency of Calindol vs. Ca2+. C, histogram of latency of effect for Ca2+ and Calindol. The latency was significantly greater for activation by Calindol (23.761.2 s) than by Ca2+ alone (9.060.2 s) in the recordings from 22 cells (p,0.001). doi:10.1371/journal.pone.0008563.g005 pocket [35,41]. Since Calindol binds to a membranous pocket [36], we asked whether the effects of Calindol on synaptosomes were mediated via mGluR. Using mGluR1-expressing HEK cells (kind gift of Dr J. Saugstad) we tested if Calindol also activated mGluR. Bath application of 10 mM glutamate increased F/F0 on average by ,4 (red trace is average of 30 cells each represented by black trace) in the mGluR1 cells (Figure 6). However, the same cells did not respond to Calindol (5 mM) when applied 10 minutes later in the presence of 1 mM [Ca2+]o. The absence of effect was not due to receptor desensitization or depletion of Ca2+ from intracellular stores as glutamate evoked a similar response when reapplied after an additional 10 minutes. Untransfected HEK cells did not respond to glutamate or Calindol (data not shown). These data strongly support the proposal that the [Ca2+] - o modulated NSCC signaling pathway in nerve terminals involves CaSR. In addition, the distinct kinetics of action for Calindol and Ca2+ confirm different mechanisms of action for these two classes of CaSR agonist. CaSR Regulates Nerve Terminals Figure 6. Glutamate (10 mM) but not Calindol (5 mM) activated the mGluR1 expressed in HEK cells. Application of glutamate (horizontal bars) caused a transient increase in fluorescence (F) relative to basal level (F0) indicating an increase in [Ca2+]i (left). The black curves denote signals from 35 cells while red curves indicate averages. Applications of test solutions were staggered by 10 min to allow for recovery. Calindol application (horizontal bar) to the same cells did not produce any change in F/F0 (middle), yet these cells remained responsive to glutamate (right). doi:10.1371/journal.pone.0008563.g006 Nerve Terminal [Ca2+]o Detector Not Modulated by Glutamate or GABA Initially, CaSR, mGluR and GABABR were all possible candidates for the neocortical [Ca2+]o sensor as they are sensitive to [Ca2+]o [15,16,17,18] and localized to the synapses of central neurons [19,20,21,22,23]. Consequently, we addressed if mGluR or GA- BABR were mediating part of the response of the terminals to changes in [Ca2+] . We addressed this issue by testing whether glutamate or o GABA modulate the NSCC currents in isolated rat neocortical nerve terminals. In cell-attached recordings, depolarization from 240 to 110 mV activated outward currents through NSCCs that increased as bath [Ca2+] was reduced (Figure 7). Co-application of 100 mM glutamate did not affect the amplitude or rate of activation of the NSCC currents (Figure 7A, red trace). In addition, glutamate did not appreciably slow the response of the terminal to changes in [Ca2+]o which is illustrated in the plot of NSCC current amplitude versus time (Figure 7B). The absence of an effect of glutamate was confirmed in recordings from 11 terminals (Figure 7C). Brain extracellular glutamate concentration varies between 25 nM at rest [42] and 1–5 mM following exocytosis [43]. While these higher levels are short-lived and thus less likely to affect GPCR signaling, we tested whether they could impact the NSCC current in nerve terminals. Glutamate (2 mM) was ineffective at altering the size or kinetics of the Ca-modulated currents in rat terminals (n = 5; Figure S3). We also tested the action of GABA (100 mM) in a parallel set of experiments (n = 3). GABA was equally ineffective at altering current amplitude, activation or responsiveness to [Ca2+]o (Figure 7D–F). The absence of any effect of glutamate or GABA effectively rules out the mGluR and GABAB receptor as candidate [Ca2+]o sensors that modulate NSCC currents in small neocortical terminals. Discussion Sustained decreases in [Ca2+]o will depress subsequent synaptic transmission due to the exquisite sensitivity of transmitter release PLoS ONE | www.plosone.org 6 January 2010 | Volume 5 | Issue 1 | e8563