Figure 3. DPKQDFMRFamide reversibly increases amplitude of evoked Ca2+ responses in nerve terminals. A, The upper images are of an OGB-loaded type 1b nerve terminal at time = 0 [stimulation (stim)], 0.25, and 0.4 s. The red arrow and white lines indicate the location of the line scans through the nerve terminal. The lower image is a composite of all of the line scan images through the same location of the nerve terminal. Aii, The fluorescence of the sequential line scans is graphed below the composite image. Decay of the calcium transient response was fit with a single exponential using the equation y = ae−bx (red line). Scale to the right of the scan represents increasing fluorescence intensity. Red arrow indicates time of stimulation. B, Increasing extracellular [Ca2+] increases amplitude of single impulse Ca2+ responses. Normalized amplitudes of single impulse Ca2+ responses increased significantly (p = 0.01; n = 6) with progressively increasing extracellular Ca2+ concentration (open triangles): 0.138 ± 0.010 at 1.25 mm Ca2+, 0.160 ± 0.015 at 1.5 mm Ca2+, 0.176 ± 0.012 at 2.0 mm Ca2+, and + was 0.194 ± 0.019 at 4.0 mm Ca2+. The time constant of decay recorded in preparations bathed in 1.25 mm [Ca2+]e was 46.2 ± 6.1 ms and was not significantly different when recorded in 4.0 mm [Ca2+]e, which was 54.3 ± 6.5 ms. C, DPKQDFMRFamide reversibly increases the amplitude of evoked calcium responses in presynaptic terminals. Ci, At 1.25 mm extracellular Ca2+, amplitude of the normalized evoked response significantly increased after peptide application (p < 0.05; n = 10). Washout (wash) of the peptide restored the amplitude, which was significantly less than with the peptide (p < 0.05). Cii, In 2.0 mm extracellular Ca2+, the normalized amplitude of the evoked response increased significantly with peptide application (p < 0.05; n = 10) and reversed with washout of the peptide (p > 0.05). Ciii, Decay time constant was not altered by the peptide at 1.25 mm Ca2+ (p = 0.8). Civ, Decay time constant was not altered by the peptide at 2.0 mm Ca2+ (p = 0.9). ctrl, Control. D, DPKQDFMRFamide does not alter steady-state free intracellular [Ca2+]. Di, Ratiometric recordings of Fura fluorescence were made at 340 and 380 nm every 5 s for 15 s, allowing quantification of [Ca2+] at rest. Dii, No difference in resting or steady-state calcium levels is detected between control and DPKQDFMRFamide-exposed nerve terminals (n = 4; p > 0.6). pre, Pre-DPKQDFMRFamide. Asterisks (*) indicate significant difference.