Supporting Online Material for

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1 Supporting Online Material for Synaptic Integration in Tuft Dendrites of Layer 5 Pyramidal Neurons: A New Unifying Principle Matthew E. Larkum,* Thomas Nevian, Maya Sandler, Alon Polsky, Jackie Schiller* *To whom correspondence should be addressed. matthew.larkum@gmail.com (M.E.L.); jackie@tx.technion.ac.il (J.S.) Published 7 August 2009, Science 325, 756 (2009) DOI: /science This PDF file includes: Materials and Methods Figs. S1 to S4 References

2 Supporting Online Material Methods Electrophysiology. Neocortical sagittal brain slices µm thick were prepared from (36 ± 5) day old Wistar rats. Whole-cell patch-clamp recordings were performed from visually identified layer-5 pyramidal neurons using infrared (IR) Dodtgradient contrast video microscopy (Fig. S3). The extracellular solution contained (in mm) 125 NaCl, 25 NaHCO 3, 25 Glucose, 3 KCl, 1.25 NaH 2 PO 4, 2 CaCl 2, 1 MgCl 2, ph 7.4 at 35º to 36 º C. The intracellular solution contained (in mm) 115 K + -gluconate, 20 KCl, 2 Mg-ATP, 2 Na 2 -ATP, 10 Na 2 -phosphocreatine, 0.3 GTP, 10 HEPES, 0.05 Alexa 594 and biocytin (0.2%), ph 7.2. Bicuculline methiodide (1 µm) was added to the extracellular solution in some experiments. Dual whole-cell voltage recordings were performed from the soma (4-6 MΏ) and dendrites (20-30 MΏ) using Axoclamp 2A and Dagan BVC-700A amplifiers. Data was acquired with an ITC-16 board and analyzed using Igor software. Focal electrical synaptic stimulation was performed via a theta patch pipette located in close proximity to the selected tuft dendritic segment guided by the fluorescent image of the dendrite. An enhanced frequency of unitary EPSPs was evoked by local application of high osmolar external solution, consisting of normal external solution with 300 mm sucrose and 1 μm TTX added, close to the dendritic site of recording. Spontaneous and sucrose-evoked unitary EPSPs were selected according to kinetic parameters with rise times of less than 0.5 ms. Data analysis. Data analysis was performed using Igor software and Excel. All statistical analysis used the Student's t-test. The amplitudes of local spikes were determined from threshold (turning point of rising phase). For computing the separation distal versus proximal electrogenesis we used Fisher s Exact test with a 2x2 contingency table comparing the outcomes Ca 2+ spike with no Ca 2+ spike and the categories distal versus proximal. Imaging. Two-photon excitation fluorescence microscopy was combined with IR- SGC(1) using a femtosecond infrared laser source (λ = 820 nm) coupled to a laser scanning microscope(2) equipped with a 40x water immersion objective (NA 0.8). IR- SGC images were generated by spatially filtering the forward scattered IR laser light with

3 a Dodt-tube and subsequent detection by a photomultiplier tube connected to one detection channel of the laser scanning microscope. Neurons were filled via the somatic or dendritic electrodes with Alexa 594 to visualize the tuft dendrites. Fluorescence and IR-SGC images were acquired simultaneously and could be overlaid online resulting in a full-frame rate of 5 Hz (Fig. S3). Glutamate Uncaging. MNI-glutamate(3), was delivered locally to a dendrite using pressure ejection (1-10 mbar) from an electrode (10 µm diameter, containing 5-10 mm caged glutamate, placed µm below the surface level of the slice and typically >18 µm lateral to the recording electrode to avoid movements of the recording electrode by the pressure. MNIglutamate was photolysed by 4 µsec pulses of a 361 nm UV-laser beam using point scan mode and the level of activation of glutamate receptors was controlled by increasing the intensity of the UV-laser. Using caged fluorecein we estimated the size of the activated dendrite to be ~5 µm which would typically contain ~5 spines However caged glutamate also activates the dendritic shaft and hence the evoked response may exceed the current due to only that many spines. Cells were imaged with a 60X, 0.9 NA water objective and a confocal system on an upright BX51WI microscope. Computer simulations Computer simulations were performed on biocytin filled and reconstructed cells used in the experimental study. Multi-compartmental ( dendrites, 11 segments for each compartment, average segment length of 10 μm) models were simulated in NEURON platform (time step 0.1 ms). The passive and active parameters were modified from (4-7). Our experimental data (sub and supra threshold current injections, pharmacological manipulations, UV laser uncaging and synaptic stimulations) introduced strong constraints to the passive and active parameters used in our model. The values for R i and R m were Ωcm and 20 kωcm 2, respectively. Other passive biophysical parameters of the model are as follows: E leak = -70 mv, E Na = 50 mv, E K = -87 mv and C m = 1.2 μfcm -2. Active conductances were distributed as follows: Axon: HH-like sodium channel ps/µm 2 ; HH-like potassium channel 8000 ps/µm 2 ; K DR 1000 ps/µm 2. Soma: HH-like sodium channel 50 ps/µm 2 ; HH-like potassium

4 channel 600 ps/µm 2 ; K DR 300 ps/µm 2 ; K A 600 ps/µm 2 ; K M 10 ps/µm 2. The apical trunk: HH-like sodium channel 50 ps/µm 2 ; Ca L 1 ps/µm 2 ; HH-like potassium channel 600 ps/µm 2 ; K DR 300 ps/µm 2 ; K A 600 ps/µm 2 ; K Ca 1 ps/µm 2. Apical tuft dendrites and basal dendrites: HH-like sodium channel 20 ps/µm 2 ; Ca L 1-3 ps/µm 2 ; K DR 2-10 ps/um 2 ; K A ps/µm 2 ; K Ca 10-50pS/µm 2. I H was inserted in the tuft dendrites with 10pS/µm 2 conductance. Apical sodium channels had a 10mV increase in the activation and deactivation kinetics. Apical hot zone was set to be located 500 μm from the soma and extend for 300 μm to the tuft region. Sodium and calcium conductances were increased to 100 ps/µm 2 and 10 ps/µm 2 respectively. To examine the effect of homogeneous voltage gated calcium distribution in the apical tree, both the tuft and hot zone Ca L conductance was set to 5 ps/µm 2. Kinetics of the NMDA current was modelled as follows: g NMDA =g MAX (e -t/70 -e -t/3 )/(1+0.3e v ). AMPA synapses were modelled with an instantaneous rise time and decay time constant of 0.5ms. The synaptic conductance of a single excitatory input had a 0.5nS AMPA and 1nS NMDA components. In part of the simulations, synapses were randomly distributed on pre-selected branches. Triple pulse stimulation with 20 ms inter-stimulusintervals was used in all synaptic activations. Spine number for every dendrite was based on direct spine counting from our recorded cells (Larkum, unpublished data). Activation of inputs on modelled dendritic spines changed the local voltage traces by less than 1%; therefore spines were not explicitly modelled in the simulations used to create the final figures.

5 Fig. S1 Attenuation of subthreshold steady-state and suprathreshold activity in the tuft. (A) Reconstruction of biocytin-filled layer 5 pyramidal neuron showing the positions of the recording electrodes 790 µm (blue) and 930 µm (red) from the soma. (B) Short EPSP-waveform current injection produced a broad potential resembling a Ca 2+ spike with injection at the proximal tuft electrode (blue) which propagated well into the distal tuft (red). (C) Distal current injection (right column) failed to initiate a spike. Current injected is indicated below in the colour of the corresponding injection electrode. (D) Long (1 s) step current injection at the proximal tuft electrode. (E) Long current injection at the distal tuft electrode. (F to G) Hyperpolarizing long current injection. (H) Attenuation of steady-state hyperpolarizing pulses away from (blue) and towards (red) the soma as a function of the separation between the two dendritic electrodes (always distal to the main bifurcation). (I) Attenuation of active events away from (blue, Ca 2+ spikes) and towards (red, Na + spikes).

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7 Fig. S2 Neuron Simulation of spiking activity and synaptic interactions in the apical tree of layer 5 pyramidal neuron. (A) Reconstruction of the cell shown in Fig. 3E-G. Calcium spike initiation zone is marked in black bold. Recording locations are marked by colored circles. (B) Simulated current injection to the distal tuft (top 947µm, 0.5nA), proximal tuft (middle 840µm, 1nA) locations and to the soma (bottom). Color coding as in (A). (C) Simulation of an NMDA spike (bottom), overlaid on an experimentally recorded one (top). The dotted line represents the local simulated voltage at the stimulation site. (D) Voltage clamp recordings at a tuft branch 950 µm from soma. All recordings were made from the same dendrite. Upper left, NMDA spike evoked by UV laser uncaging. The recording was performed in current clamp mode to define the subthreshold (black trace) and suprathreshold (red trace) UV laser intensity that is needed for NMDA spike initiation. Uncaging was performed 10 µm distal to the recording site nearby the sucrose pipette. Upper right, same as upper left, in voltage clamp mode in order to measure the threshold current (black trace) for NMDA spike initiation. Upper right and lower right present the simulation of the corresponding experimental data. (E to F) Simulated NMDA spike amplitude, as recorded at the calcium spike initiation zone, decrease exponentially with distance from pia. (E). Similarly, a decrease in the number of synapses required to initiate an NMDA spike with distance from pia is seen (F). Voltage gated calcium channel (VGCC) concentrations have only a small effect on distal NMDA spike amplitude and the number of synapses needed to initiate a spike. (No tuft VGCC, black circles; tuft VGCC concentration of 1pS/µm 2 (as used in B to C), opaque squares; VGCC concentration of 3pS/µm 2, grey circles). (G to H) Example traces of synapse activation in 3rd order branches when NMDA spike initiate (G) and when AMPA-only synapses are activated (H). Color coding as in (A to C). Activation of 100 synapses is sufficient to trigger NMDA spike at the second pulse and calcium spike at the third (G). Similar number of AMP-only synapses leads to small subthreshold EPSPs (H, left). A fivefold increase in the number of synapses was needed to generate a calcium spike near the main bifurcation (H, right ).

8 Fig. S3 Experimental setup for fluorescence and IR-scanning gradient contrast (IR-SGC) guided patch clamp recordings from distal tuft dendrites. (A) Schematic representation of the experimental setup. The same light path that is used to visualize the brain slice with IR-gradient contrast video microscopy (flip mirror in, moveable mirror out) is used in the reverse direction during 2-photon fluorescence imaging (flip mirror out, moveable mirror in) to generate an equivalent IR-image. The forward scattered IR-laser light is collected by the condenser and spatially filtered by the quarter-segment aperture in the Dodt-tube and then focused onto a photomultiplier (PMT) that is attached to the acquisition electronics of the laser-scanning unit. The simultaneously acquired 2-photon fluorescence image can be overlaid with the IR-SGC image online allowing visual guidance of the patch-clamp electrodes towards the dendritic branches. IR and fluorescence light are separated by a dichroic mirror (670DCXXR). In the fluorescence detection pathway below the condenser IR-laser light is blocked by an IR-filter (E700SP) and a dichroic mirror (560DCXR) together with bandpass filters allows the separation of red (HQ630/60) and green (HQ525/50) fluorescence. (B) Example images of the IR-SGC channel (left), the red fluorescence channel (middle) and the overlay (right) for a distal tuft branch (upper row, 862 µm from the soma) and the main apical branch at the first bifurcation (lower row, 630 µm from the soma). L5 pyramidal neuron somata or proximal main apical dendrites were initially visualized using IR-gradient contrast video microscopy and filled with Alexa594 by whole-cell recordings. The filling electrode was retracted and the imaging mode was switched to laser-scanning resulting in the visualization of the Alexa594-stained dendritic arborization simultaneously with the IR-SGC.

9 The fluorescence image facilitated the identification of the selected dendritic branch in the IR-SGC image, which was mainly used to establish the dendritic recording by standard procedures. Fig. S4 Dendritic spike generation correlates with dendritic diameter. (A) The diameter of a tuft branch decreases with each bifurcation (branch order) in daughter branches. Branch order 1 denotes the segment from and including the major apical bifurcation. A significant drop in dendritic diameter (**, p < 0.01) was only found for branch order 2, after which dendritic diameter decreased only gradually with subsequent branching. (B) Histogram of the size distribution of dendrites that were capable (blue) or incapable (red, i.e. either generated a Na + spike or no spike) of generating Ca 2+ spikes. (C) Comparison of the average diameter of dendrites that responded with a Ca 2+ spike or did not initiate a Ca2 + spike to current injection. Dendrites that generated a Ca 2+ spike were significantly larger than dendrites that did not generate a Ca 2+ spike (**, p < 0.01). (D) Threshold of Na + spikelets (solid, red) and Ca 2+ spikes (open, blue) according to branch order (legend) and dendrite diameter. (E) Left, a histogram showing the range of Na + spikelet amplitudes measured at distal tuft dendrites; right, three examples of sodium spikes before (red) and after TTX (black) showing small, medium and large size spikelets.

10 Reference List 1. V. C. Wimmer, T. Nevian, T. Kuner, Pflugers Arch. 449, 319 (2004). 2. J. Rathenberg, T. Nevian, V. Witzemann, J. Neurosci Methods 126, 91 (2003). 3. M. Matsuzaki et al., Nat. Neurosci. 4, 1086 (2001). 4. P. A. Rhodes, R. R. Llinas, J. Physiol 536, 167 (2001). 5. A. T. Schaefer, M. E. Larkum, B. Sakmann, A. Roth, Journal of Neurophysiology 89, 3143 (2003). 6. M. Migliore, L. Messineo, M. Ferrante, J. Comput. Neurosci 16, 5 (2004). 7. T. Nevian, M. E. Larkum, A. Polsky, J. Schiller, Nat. Neurosci. (2007).