Article Figures & Data
Figures
- Figure 1.
HCRT induces somatodendritic release from LC neurons. A, One hundred nanomolar HCRT did not evoke a significant amperometric signal (Iamp). Normal or high-K ACSF was applied as a negative (Control) or positive control, respectively (n = 6). B, In contrast to 100 nm HCRT, 1 μm HCRT markedly elicited secretion (p < 0.01; n = 9). The inset shows that 1 μm, but not 100 nm HCRT, increased the action potential firing frequency and induced depolarization (11.6 ± 1.8 mV; n = 5). C, One micromolar HCRT did not induce an amperometric response in the presence of SB 334867 (1 μm). Inset, Statistical data of the integral of amperometric current (HCRT, 4.7 ± 0.9 pC; HCRT+SB, 0.31 ± 0.06 pC; Recovery, 3.0 ± 1.1 pC; n = 5). D, After clearing the extracellular matrix from the soma surface using a cleaning pipette, amperometric spikes induced by high-K or 1 μm HCRT were detected (n = 5). Asterisks indicate single typical amperometric spikes as expanded in insets. *p < 0.05 and ***p < 0.0001 in this and the following figures. Error bars indicate SEM.
- Figure 2.
NMDA evokes somatodendritic catecholamine release in LC neurons. A, One hundred micromolar NMDA elicited a notable Iamp. Right, The normalized amount of secretion caused by high K (24.9 ± 7.3 pC), ACSF (Control; 2.4 ± 0.9 pC), or 100 μm NMDA (9.4 ± 2.3 pC) (n = 10). B, NMDA dose-dependently evoked secretory responses. Right, The normalized secretion induced by NMDA applied at concentrations of 1 (3.9 ± 1.0 pC), 20 (5.7 ± 1.2 pC), and 100 μm (7.1 ± 1.8 pC) (n = 14). Inset, Treatment with the NMDA receptor antagonist APV (50 μm) blocked the 100 μm NMDA-induced secretion (n = 5).
- Figure 3.
HCRT potentiates NMDAR-mediated somatic secretion through activation of Hcrtr 1 and PKC in LC neurons. A, Examples of Iamp (top traces) and integrated current signals (∫Iampdt) evoked by 100 μm NMDA before and after application of 100 nm HCRT for 5 min. B, Time course of 100 nm HCRT or ACSF (Control) action on NMDA-induced secretion. Amperometric signals were elicited every 5 min by 100 μm NMDA before and after HCRT treatment (n = 9). C, In the presence of SB 334867 (1 μm), 100 nm HCRT did not increase the NMDA-induced secretion. D, HCRT-mediated increase in NMDA-induced secretion was blocked by pretreatment with the PKC inhibitor BIS II (1 μm) for 45 min. E, One micromolar PMA significantly increased NMDA-induced secretion in a manner similar to HCRT. F, Bar graph summarizes the statistical data of the normalized amount of secretion in C [control (Con), 8.2 ± 2.1 pC; HCRT+SB, 7.8 ± 1.9 pC; p > 0.05; n = 5], D (Con, 9.8 ± 2.7 pC; HCRT+BIS, 9.3 ± 2.9 pC; p > 0.05; n = 5), and E (Con, 18.0 ± 6.1 pC; PMA, 27.3 ± 9.5 pC; p < 0.05; n = 4).
- Figure 4.
HCRT potentiates NMDAR-mediated [Ca2+]i elevation via activation of Hcrtr 1 and PKC in LC neurons. A, NMDA- or HCRT-induced secretion was dependent on extracellular Ca2+. Application of Ca2+-free ACSF containing 1 mm EGTA and 200 μm CdCl2 for at least 30 min significantly blocked secretion induced by 100 μm NMDA (top traces; n = 4) or 1 μm HCRT (bottom traces; n = 5). After adding normal ACSF containing 2.5 mm Ca2+ to the perfusion solution, the evoked amperometric signals recovered. B, Left, Representative image of a fura-2-loaded LC neuron under bright-field DIC (left) or fluorescence (F380; right) conditions. A black box over the soma of this neuron indicates the region of interest. Right, One micromolar, but not 100 nm, HCRT significantly increased [Ca2+]i (n = 4). C, Application of 100 nm HCRT for 5 min increased the 100 μm NMDA-induced calcium response. D, E, The HCRT-mediated increase in NMDA-elevated [Ca2+]i was blocked by SB 334867 (1 μm) or BIS II (1 μm). F, Bar graph summarizes the statistical data of normalized changes in F340/F380 ratios in C [control (Con), 0.026 ± 0.007 arbitrary units; HCRT, 0.039 ± 0.009; p < 0.05; n = 9), D (Con, 0.04 ± 0.01; HCRT+SB, 0.033 ± 0.009; p > 0.05; n = 5) and E (Con, 0.02 ± 0.007; HCRT+BIS, 0.017 ± 0.007; p > 0.05; n = 6).
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