P/Q And N Channels Control Baseline and Spike-Triggered Calcium Levels in Neocortical Axons And Synaptic Boutons Yuguo Yu, Carlos Maureira, Xiuxin Liu and David McCormick Supplemental Figures 1-9 1
Figure S1. Properties of OGB1 on synaptic transmission and axonal imaging. a. Oregon green Bapta 1 at the concentrations used in this study does not disrupt the ability of depolarization of the presynaptic somatic membrane potential to facilitate synaptic transmission between pairs of layer 5 pyramidal cells. Control data illustrating the shift in single EPSP amplitude evoked by single action potentials between nearby layer 5 pyramidal cells (n=4 pairs). Red is with the presynaptic cell depolarized to -60 mv, while blue is with the presynaptic cell at -80 mv. b. Similar data, except with 50 μm OGB1 in the micropipettes (n=4 pairs). c. Photograph of the lower part of the soma and axon initial segment as seen in the 2-photon microscope. The green box illustrates the area in which data was collected in both the green (OGB1) and red (Alexa Fluor 594) channels. d. Raw fluorescence in the green (Oregon Green Bapta) channel versus distance from the beginning of the axon, at -80 and -60 mv. Data is plotted on a log plot, owing to the large somatic signal. e. Subtraction of the -80 mv trace from the -60 mv trace reveals that subthreshold depolarization resulted in an increase in Ca 2+ concentration in the axon initial segment, but not the soma. 2
Figure S2. Relationship between action potential activity and OGB1 and fluo 5F fluorescence. a. OGB1 fluorescent response in the AIS to 1, 2, 3, 12 and after 50 action potentials. Saturation was considered to be the response to 50 action potentials. a. The fluorescent response to 1, 2, and 3 spikes at 50 Hz was approximately linear. b. Plot of the ratio between Fpeak and F showing a F/F of 2.25. Similar numbers were obtained in group (n=6) averages from different compartments: OGB1: soma: 2.2+-0.2; AIS: 2.2+-0.15; boutons: 2.1+-0.1. c. Plot of ΔF/R for OGB1 in the AIS for the data obtained in b. For the first 3 action potentials, ΔF/R is approximately 30% for each spike. d. Fluo5F fluorescent response to 1, 2, 3, 12 and after 50 action potentials at 50 Hz in the AIS. Saturation was considered to be the response to 50 action potentials. The fluorescent response to 1, 2, and 3 spikes was approximately linear. e. Plot of the ratio between Fpeak and F showing a F/F of 8.06. Similar numbers were obtained in group (n=6) averages fluo5f:soma: 8.9+-1.6; AIS: 7.4+-1.5; boutons: 8.1+-1.6. f. Plot of ΔF/R for the data obtained in b for the AIS. For the first three spikes, each action potential initiates a change in ΔF/R of about 140%. 3
Figure S3. Comparison of depolarization induced enhancement of calcium responses in the axon initial segment and proximal boutons obtained with Fluo 5f at 21 and 35 o C. Significant enhancements (asterisks) were found for all cases. There were no statistically significant differences between the results obtained at 21 and 35 o C and therefore the results were combined (middle bars). a) Changes in baseline Fluo 5f response to depolarization. b) change in peak Fluo 5f signal obtained after an action potential. c) Depolarization induced change in delta peak Fluo 5f change after an action potential. 4
Figure S4. Bath application of NiCl 2 blocks the depolarization-induced and spike-triggered increases in somatic and axonal Ca 2+. a-d. Bath application of Ni 2+ (200 μm) results in a large reduction or abolition of the effect of membrane potential on basal Ca 2+ levels (a-c) or spike triggered Ca 2+ increases (a,d). 5
Figure S5. Bath application of 100 μm of NNC 55-0396 (Huang et al., 2004) does not block the depolarization-induced and spike-triggered increases in Ca 2+ in the axon initial segment. a-c. This t-current antagonist, which has some specificity towards Cav3.1 subunits, did not exhibit any significant effect (10-100 μm; n=26 observations in 5 cells). d. NNC application did not have an effect on the spike triggered increase in Ca 2+ in the axon initial segment. 6
Figure S6. Effects of the T-current blocker mibefradil. Bath application of mibefradil (30 μm) (Martin et al., 2000) enhanced the depolarization-induced increase in baseline Ca 2+ (a-c) but had no significant effect on spike-triggered Ca 2+ transients in the axon initial segment (d) (3-30 μm; n=76 observations in 7 cells). 7
Figure S7. Bath application of the T-current blocker fluoxetine does not affect axonal calcium responses. Application of 50 μm of fluoxetine, an antagonist of all three types of T-channel alpha subunits (Cav3.1, 3.2, 3.3) (Traboulsie et al., 2006) was without effect on the depolarization-induced or spike triggered increases in Ca 2+ in the axon initial segment (50 μm; n=6 observations in 2 cells). 8
Figure S8. Effects of SNX-482 on calcium responses in the axon initial segment. Bath application of the Ca 2+ channel antagonist SNX-482 (500 nm; n=21 observations in 4 cells) resulted in no significant effect on the depolarization-induced increase in Ca 2+ in the axon initial segment, although there was a small but significant decrease 12+-8.6% in the spike-triggered Ca 2+ response (d). SNX-482 is reported to be a selective antagonist of R-type Ca 2+ channels, although it may also inhibit P/Q channels as well (Arroyo et al., 2003). 9
Calculation of the approximate [Ca 2+ ] at and during depolarization For Oregon Green Bapta 1, the internal concentration of Ca 2+ can be approximated according to the following equations (Maravall et al., 2000; Jackson and Redman, 2003; Scott and Rusakov, 2006): For OGB1: Oregon Green BAPTA 1: Kd=200 μm [Ca] F Fmin F/ F F / F = = Kd F F 1 F/ F F min min = 6 F (approximate value) [Ca] F/ F 0.1667 => = Kd 1 F/ F [1] [2] For steady state ing calcium concentration: [Ca] F / F 0.1667 = Kd 1 F / F Intracellular ing calcium concentration ranges from: [Ca] = 80 120 nm,here we assume [Ca] = 100 nm : [Ca] 100 F / F 0.1667 0.5 Kd 200 1 F / F => = = = => F / F = 0.4444 For subthreshold depolarization increased ing [Ca] level: [Ca] x x = Cx[Ca] ; F = fxf x [Ca] x Fx / F 0.1667 = Kd 1 F / F Cx[Ca] fxf / F 0.1667 0.4444 fx 0.1667 Kd 1 fxf / F 1 0.4444 fx => = = 0.4444 fx 0.1667 => 0.5Cx = 1 0.4444fx 0.8888 fx 0.3333 => Cx = 1 0.4444 fx x [3] This equation is plotted in supplemental Figure S8 below. From this figure, we see that a 5% increase in Fluorescence intensity F, corresponds to a 12% increase in ing internal calcium concentration; while a 20% increase in Fluorescence intensity F corresponds to a 57% increase in ing internal calcium concentration. 10
If the original [Ca 2+ ] i is 100 nm, a 5% in OGB1 fluorescence, such as in the presynaptic boutons, suggests an increase in [Ca 2+ ] i to 112 nm while a 20% increase in OGB1 fluorescence, such as in the AIS, suggests an increase in [Ca 2+ ] i to 157 nm. The relationship between released transmitter and baseline calcium concentration is not well known. It has been suggested to follow a power-law relationship: ΔPSP = Δ[Ca] n with n=1.1 (Awatramani et al., 2005). If n=1.1, a 5% change in OGN1 fluorescence 12% increase in [Ca 2+ ] i 13.5% increase in average PSP amplitude. In contrast, a 20% change in OGB1 F 57% increase in [Ca 2+ ] i 65% increase in average EPSP amplitude. Since we observed changes in [Ca 2+ ] i that on average were approximately 5%, but which ranged up to approximately 20% (Figure 2), we should expect that depolarization of the soma could cause a small (e.g. 12%) increase in average EPSP amplitude, with rare increases that are up to 65% with subthreshold depolarization. Figure S9. Relationship between percent change in internal free calcium concentration and percent change in fluorescence of OGB1 indicator dye. Note that for a 5% increase in OGB1 fluorescence corresponds to a calculated 12% increase in [Ca 2+ ]free and a 20% increase in OGB1 11
fluorescence corresponds to approximately a 57% increase in [Ca 2+ ]free. The red line is the speculative amplitude of the average EPSPas a function of the change in OGB1 indicator fluorsescence, using the power law exponent of 1.1 as per (Awatramani et al., 2005). References Arroyo G, Aldea M, Fuentealba J, Albillos A, Garcia AG (2003) SNX482 selectively blocks P/Q Ca2+ channels and delays the inactivation of Na+ channels of chromaffin cells. Eur J Pharmacol 475:11-18. Awatramani GB, Price GD, Trussell LO (2005) Modulation of transmitter release by presynaptic ing potential and background calcium levels. Neuron 48:109-121. Huang L, Keyser BM, Tagmose TM, Hansen JB, Taylor JT, Zhuang H, Zhang M, Ragsdale DS, Li M (2004) NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-Nmethylamino)ethyl)-6-fluo ro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride]: a new selective inhibitor of T-type calcium channels. J Pharmacol Exp Ther 309:193-199. Jackson MB, Redman SJ (2003) Calcium dynamics, buffering, and buffer saturation in the boutons of dentate granule-cell axons in the hilus. J Neurosci 23:1612-1621. Maravall M, Mainen ZF, Sabatini BL, Svoboda K (2000) Estimating intracellular calcium concentrations and buffering without wavelength ratioing. Biophys J 78:2655-2667. Martin RL, Lee JH, Cribbs LL, Perez-Reyes E, Hanck DA (2000) Mibefradil block of cloned T- type calcium channels. J Pharmacol Exp Ther 295:302-308. Scott R, Rusakov DA (2006) Main determinants of presynaptic Ca2+ dynamics at individual mossy fiber-ca3 pyramidal cell synapses. J Neurosci 26:7071-7081. Traboulsie A, Chemin J, Kupfer E, Nargeot J, Lory P (2006) T-type calcium channels are inhibited by fluoxetine and its metabolite norfluoxetine. Mol Pharmacol 69:1963-1968. 12