Supplementary information to: Is action potential threshold lowest in the axon? Maarten H. P. Kole & Greg J. Stuart Supplementary Fig. 1 Analysis of action potential (AP) threshold criteria. (a) Example of APs from the axon initial segment (AIS, blue) and soma (black) during extracellular synaptic activation. Recordings were obtained with 10 khz analogue filtering and 50 khz sampling. (b) Voltage phase-plane projection 5 of axonal and somatic dv/dt versus membrane potential (V) of the onset of the AP. 1
Here, the voltage threshold is visible as the membrane voltage where the rate-of-change in voltage suddenly increases above the baseline noise. The black dotted line indicates three standard deviations (S.D.) of the baseline noise. The S.D. of the somatic and axonal baseline were approximately 10.4 V s 1 and 7.6 V s 1, respectively. The red dotted line indicates a threshold level of 50 V s 1. Note the shallower slope of the rising phase in the axon compared to the soma as observed recently 6. Inset shows the voltage phase-plane projection for the full range of membrane potentials. Scaling kv s 1 and mv. (c) Examination of different voltagerate criteria for voltage threshold. Example locations are shown for 10 (blue), 50 (red) and 100 V s 1 (green). Recordings were digitally filtered at 10 khz. Note that 50 V s 1 coincides best with the inflection in the somatic voltage trace. (d) Quantification of the voltage threshold difference (AIS Soma) using an action potential threshold criteria of between 10 and 100 V s 1 giving an axon to soma voltage difference of between 2.6 ± 0.8 mv and 7.7 ± 0.8 mv (n = 7). Red line is an exponential fit to the average data. Data shown as mean ± SEM. (e) A separate analysis of voltage threshold from the same recording was performed according to the method of Henze and Buzsaki 7 by taking the membrane potential at the time of the first peak in the 3 rd derivative of action potential voltage. Recordings were digitally filtered at 10 khz. Left, high magnification of the somatic AP threshold and aligned the 3 rd derivative of action potential voltage (bottom). Right, high magnification of the AIS voltage trace with 3 rd derivative of action potential voltage (bottom). Note that using this criterion to define AP threshold the voltage threshold was 10.5 mv more depolarized in the AIS compared to the soma. In four randomly chosen recordings the voltage difference at threshold using this method was on average 10.2 ± 0.8 mv (n = 4). 2
Supplementary Fig. 2 Isolation of the SD spike in the model. (a) Left, Full morphology of the layer 5 pyramidal neuron model. Right, Close-up of the somato-dendritic (SD) and axonal sections. The SD membrane had a Na + channel conductance (gna) of 60 ps µm 2 in all simulations. gna in myelinated sections was 100-fold lower than gna in the axon. (b) Control somatic voltage responses to 0.2 na increasing current steps from 0.4 to 1.2 na. Note a fast rising action potential with a low voltage threshold (red dot, 49.9 mv). (c) To mimic TTX-mediated block of axonal Na + channels gna in the entire axon was set to 0. Somatic voltage responses were triggered by current injections between 2 and 6 na. Removal of Na + channels from the axon increased voltage threshold (red dots, 23.8 and 25.9 mv) by ~25 mv, and current threshold increased ~4-fold (from 1.2 to 5 na). These data are consistent with the experimental findings during local block of Na + channels in the AIS by TTX (Fig. 3). 3
Supplementary Methods Female Wistar rats (3-4 weeks of age) were deeply anaesthetized by Isoflurane and quickly decapitated. One brain hemisphere was removed and parasagittal brain slices (300 µm thick) from somatosensory cortex prepared. Throughout the slicing preparation the brain was maintained in ice-cold ACSF consisting of (in mm): 125 NaCl, 25 NaHCO 3, 3 KCl, 1.25 NaH 2 PO 4, 25 glucose, 2 CaCl 2 and 7 MgCl 2 (ph 7.4; oxygenated with 95% O 2 / 5% CO 2 ). After cutting, slices were transferred to a holding chamber filled with oxygenated ACSF maintained at 35 C for 45 min, and thereafter stored at room temperature. Brain slices were transferred to an upright microscope equipped with IR-DIC optics (Olympus BX 51WI, Olympus, Japan). The microscope bath was perfused with oxygenated (95% O 2, 5% CO 2 ) ACSF consisting of (in mm): 125 NaCl, 25 NaHCO 3, 3 KCl, 1.25 NaH 2 PO 4, 25 glucose, 2 CaCl 2 and 1 MgCl 2. Dual current-clamp whole-cell recordings were made from the soma and apical dendrite 1 or axon 2 of large layer 5 pyramidal neurons using identical Dagan BVC-700A amplifiers (Dagan Corporation, Minneapolis, MN). Whole-cell patch pipettes were made from borosilicate glass (Harvard, Edenbridge, Kent, UK) pulled to an open tip resistance of 5 MΩ (soma and AIS blebs) or 10 12 MΩ (intact AIS and dendrite). Pipettes were filled with (in mm) 110 K-Gluconate, 20 KCl, 4 Mg-ATP, 0.3 Na-GTP, 10 HEPES and 10 Na 2 - Phosphocreatine (ph 7.4 with KOH; Osmolarity adjusted to 280 mosmol). Unless otherwise stated all chemicals were obtained from Sigma-Aldrich (Sigma-Aldrich Inc., St. Louis, MO). The axonal and dendritic recording distances were taken as linear estimations from the IR- DIC image, providing accurate distance estimations 2. Voltage was analogue low-pass filtered at 10-30 khz and digitally sampled at 50-100 khz using an A-D converter (ITC-18, Instrutech, USA) and data acquisition software Axograph X (v1.1.4, Axograph Scientific, Sydney, Australia). The access resistance (soma < 20 MΩ, axon or dendrites < 40 MΩ) was fully compensated by adjusting the bridge balance and capacitance neutralization. Full capacitance compensation was essential to allow temporal separation of the fast dv/dt changes during the rising phase of the AP, which in the axon of layer 5 neurons can peak at values ~1,200 V s 1 at 35 ºC. Errors in bridge balance of the somatic electrode were small (< 1 mv) when using current injections of ~1 na (n = 3; experiments with double somatic whole-cell recording). Synaptic stimulation (Fig. 1) was performed using a patch pipette filled with ACSF placed near the apical dendrite ~200 µm from the soma. Simulated EPSPs (Fig. 2) were generated by current injection through the somatic electrode with an exponential rise time of 0.2 ms and decay of 2 ms 3. Focal TTX (Tocris, Bristol, UK) application to the axon initial segment (AIS, Fig. 3) was performed with a patch pipette containing ACSF plus 0.5 1 4
µm TTX connected to a pressure-controlled device (Picospritzer III, Intracel LTD, Herts, UK). The spreading radius of the puffed solution under these conditions has previously been determined to be 30 50 µm (see Supplementary Fig. S3 in Kole et al., 2007 2 ). Voltages were not corrected for the liquid junction potential (+12 mv). Unless otherwise stated voltage threshold was defined as the membrane potential at which the rate-of-rise crosses 50 V s 1, which is greater than 4 times the S.D. of the noise (Supplemental Fig 1). Current threshold was defined as the minimum current required to evoke an AP, and assessed using brief current pulses (3 ms) into the soma or AIS. AP threshold in the AIS measured in sealed-end bleb structures ( 42.5 mv, n = 13) was similar to that observed in recordings from intact axons ( 43.8 mv, n = 13, P > 0.5). The compartmental model morphology, properties and distribution of voltage-dependent K +, Na + and HCN channels was as described previously 4. Our standard model consisted of a 3D reconstructed soma, dendrites and AIS of a layer 5 pyramidal cell with an artificial axon attached at the end of the AIS consisting of alternating sections of myelinated axon (diameter and length: 1.6 µm and 60 µm, respectively) and nodes of Ranvier (diameter and length: 1.1 µm and 1 µm, respectively). Unless otherwise stated, Na + channels were inserted into the soma, dendrites and axon using models with kinetics and voltage dependence as previously described 4. In brief, the voltage-dependence of activation and inactivation of Na + channels in the AIS and axon was 10 and 6 mv, respectively, more hyperpolarized than that of somatic and dendritic Na + channels. With this hyperpolarized voltage-dependence the absolute voltage threshold shifted about 15 mv and current threshold lowered by 2-fold. Na + channel density was set to 60 ps µm 2 in the soma and dendrites, and 3,000 ps µm 2 in the AIS and nodes of Ranvier 4 (unless otherwise stated). 1. Davie, J.T., et al. Nat Protoc 1, 1235-1247 (2006). 2. Kole, M.H.P., Letzkus, J.J. & Stuart, G.J. Neuron 55, 633-647 (2007). 3. Hausser, M. & Roth, A. J Neurosci 17, 7606-7625 (1997). 4. Kole, M.H.P., et al. Nat Neurosci 11, 178-186 (2008). 5. Jenerick, H. Biophys J 3, 363-377 (1963). 6. McCormick, D.A., Shu, Y. & Yu, Y. Nature 445, E1-E2 (2007). 7. Henze, D.A. & Buzsaki, G. Neuroscience 105, 121-130 (2001). 5