1. Volume 89, Issue 4, Pages 770-783 (February 2016)
Axonal Filtering Allows Reliable Output during Dendritic Plateau-Driven Complex Spiking in CA1 Neurons 
Pierre F. Apostolides, Aaron D. Milstein, Christine Grienberger, Katie C. Bittner, Jeffrey C. Magee 
Neuron 
Volume 89, Issue 4, Pages (February 2016)
DOI: /j.neuron
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1
The Majority of Somatically Recorded APs Reliably Forward-Propagate in the Axon
(A) 2-photon maximal intensity z stack of a typical dual recording from the soma and axon of a CA1 neuron. The axon recording location (373 μm from soma) is denoted by green pipette symbol.
(B) Example traces from the neuron in (A) showing axonal and somatic APs (upper and middle traces, respectively) during 100 ms current steps of increasing amplitude. The lower traces are the dV/dt of the somatic voltage, highlighting the dramatic reduction in the AP dV/dt due to inactivation of Na+ channels.
(C) Detail of the red rectangle in (B). The dotted lines connect axonal APs with the somatic dV/dt trace. Of note is that even the smallest APs propagate in the axon, highlighting the extensive variability in the somatic signature of axonal APs. The asterisks denote a somatic event that did not occur with an AP in the axon, likely reflecting the depolarization-induced separation of axonal and somatic AP components.
(D) Summary showing somatic dV/dt plotted against the repolarization voltage of individual APs elicited during n = 9 experiments similar to (A)–(C). The red points denote somatic APs that occurred in absence of a concomitant axonal AP. Of note is that these non-axonal APs have the lowest dV/dts and selectively occur at elevated membrane potentials.
(E) Axon spike probability as a function of peak dV/dt. The 95% cutoff values were determined by fitting the data with a sigmoid function of y = 1/(1+exp((xhalf-x)/rate)).
(F) Axon spike probability as a function of repolarization voltage. The legend is the same as (H). A sigmoid function: y = 1+{-1/(1+exp((xhalf-x)/rate))} was used.
(G) Example complex spikes recorded in an awake, head-fixed mouse running on a linear treadmill. The lower trace is the dV/dt of the somatic voltage. The peaks and troughs are cut at the dotted lines to emphasize the slowly rising events. The events highlighted in red were predicted as non-axonal APs, for the logistic regression model classified these as having a <95% probability of originating in the axon. Of note is the resemblance to the delayed somatodendritic AP component occurring shortly after an axon AP (e.g., asterisks in C). The mean AP frequency during these complex spikes was 113, 182, and 143 Hz, consistent with the notion that complex spikes represent epochs of the shortest inter-spike intervals in CA1 neurons (Harris et al., 2001; Bittner et al., 2015).
(H) Repolarization voltage and peak dV/dt for individual APs recorded during complex spikes in vivo are plotted on the x and y axes, respectively. The data are 2,129 APs from n = 10 cells. The red points are APs falling below the 95% cutoff in the logistic regression model and thus predicted as non-axonal.
(I) Instantaneous frequency for APs 2–8 plotted as a function of position in the complex spike burst. The gray dashed lines represent individual neurons and red points are mean ± SEM.
(J) Fraction of predicted axonal APs plotted against AP position in the burst. The color scheme is the same as (I).
Neuron  , DOI: ( /j.neuron )
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3. Figure 2 Spatial Profile of AP Amplitude within the Axon
(A) Experimental setup for (B)–(D). Whole cell recordings are obtained from the soma; an extracellular pipette is used to sequentially record the extracellular AP waveform at various points along the proximal axon.
(B) Z stack from a representative neuron showing the extracellular axon electrode near the proximal axon.
(C) Example traces from the neuron shown in (B). The black trace is the averaged intracellular AP waveform. The gray traces are averages of the extracellular AP waveform recorded at the soma or at three different sites along the proximal axon. The black dots on the extracellular waveforms signify the onset latency of the extracellular AP, defined as the time that the waveform reaches 10% of its maximum height. The red dotted lines show the onset latency of the AP recorded at the soma and at the point of the longest delay recorded in the axon (27 μm). Onset at the two other locations (13 and 38 μm) occurs prior to the extracellular somatic waveform, but with a shorter delay than at the 27 μm location.
(D) Summary data from n = 6 experiments similar to (B) and (C). The lines connect individual experiments. The y axis is the onset latency at different points after subtracting the onset latency of the extracellularly recorded somatic waveform (Palmer et al., 2010). The red points signify the location of longest latency. These data indicate that APs in mouse CA1 neurons initiate 27.4 ± 1.6 μm from the soma (n = 6 cells and 6–12 sites sampled per cell).
(E) Experimental setup for (F)–(I). Whole cell recordings are obtained from the soma and APs are recorded in loose-patch configuration at proximal or distal locations along the axon.
(F) Example single trial from a proximal axon recording (93 μm from soma). The lower trace shows the somatic voltage during a large square pulse current step, and the upper trace shows APs simultaneously recorded in the axon. Of note is that both soma and axon AP amplitudes rapidly decrease during the train. The black and gray arrows denote the first and fourth APs evoked during the burst. The axon AP triggered averages of somatic and axonal waveforms during 11 trials similar to the left panel are shown (right). The first and fourth APs are overlaid. The color code is the same as the arrows.
(G) Same as (F), but for a distal axon recording (373 μm from soma; data are from the same cell shown in Figures 1A–1C). Of note is the lack of co-variation in the amplitude of the axon and soma APs.
(H) Summary showing the relative timing of axon and soma APs increasing as a function of distance from the soma for full-amplitude APs and small spikelets (black and gray symbols, respectively). The data points are from n = 18 and n = 16 cells for full-amplitude APs and spikelets (2/18 cells did not reliably generate small spikelets during current steps). The dotted red line indicates t = 0, highlighting that APs recorded in the proximal axon typically occurred prior to soma AP peak during full amplitude APs as well as spikelets.
(I) Summary data (n = 18 cells) plotting the degree of AP amplitude attenuation (defined as the amplitude ratio of the fourth and first axon APs) against the distance of the axon recording electrode from the soma. The data are fit with a sigmoid function yielding a half-maximal x-value of 129 μm (red).
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

4. Figure 3
Plateau Potentials Do Not Increase Ca2+ Influx beyond the Proximal Axon
(A) Example cell filled with Alexa594 and the high-affinity Ca2+ indicator OGB1. The roman numerals indicate line scan locations along the axon.
(B) Upper trace: a train of five APs (black) or a current step in TTX (red; 2 nA, command current not shown) generates Ca2+ signals at the locations marked in (A). Lower traces: the fluorescence measurements during line scans at the locations denoted in (A) during APs (black) or plateau-like steps (red) are shown.
(C) Summary showing the steep distance dependence of axon Ca2+ signals during somatic current steps in TTX (red symbols, 81 locations in n = 9 cells). The data are fit by a monoexponential function yielding a λCa2+ = 21 μm (red line). Plotted on the same graph are peak fluorescence transients generated by trains of 3–5 APs at 100 Hz (black symbols, 45 line scan locations in n = 7 cells), revealing that this distance dependence is not due to a lack of Ca2+ channels in more distal axon regions. The AP data are fit with a linear function.
(D) Example neuron from which nerve terminal bouton Ca2+ transients were imaged with OGB1. The red rectangle denotes location of imaged bouton.
(E) Upper panel: z stack from the area denoted by the rectangle in (D), at higher magnification and tilted horizontally. The dotted line denotes line scan location. Lower panel: a complex spike (black) drives Ca2+ signals in the bouton. The bath application of TTX (red) abolishes the Na+ APs and spikelets as well as bouton Ca2+ signals.
(F) Summary from n = 12 boutons similar to (E). The red symbols are mean ± SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

5. Figure 4
K+ Channels Actively Limit Somatic Plateau Propagation in the Axon
(A) Example traces from the soma (black) and three distances along the axon (gray) during somatic current injections in a computational model of a CA1 neuron. Of note is the gradual recovery of AP height and after-hyperpolarizations (AHP) at the distal axon locations.
(B) Distance plot for the amplitude ratio of fourth axon AP/first axon AP in the model simulations.
(C) Example simulation traces in absence of a Na+ conductance to drive spikes. The propagation of plateau-like somatic depolarizations is significantly attenuated at 175 μm down the axon. Uniformly reducing axonal K+ conductances by 70% increases the propagation of somatic waveforms into the axon. The vertical arrows denote the degree of steady-state attenuation in the two simulation conditions. These simulations imply that axonal K+ channels actively limit the passive propagation of non-AP waveforms in the axon.
(D) Summary plot for the different simulation conditions showing distance-dependent propagation of slow somatic voltages. The open symbols are simulations with Na+ and K+ channels, as in (A). Of note is that the slow waveform is nearly absent due to activation of K+ channels that mediate the AHP and repolarize the axonal voltage during high-frequency APs. The filled symbols are simulations in absence of Na+ channels, with either 100% or 30% of axonal K+ channels (black and red symbols, respectively).
(E) Experimental setup for (F)–(I): dual whole cell recordings are obtained from the soma and the axon bleb.
(F) Example dual recordings from soma and axon bleb (134 μm from the soma). Left: in TTX, somatic current injection (300 ms, black traces) results in a plateau-like depolarization that strongly attenuates in the axon (gray traces). Blocking K+ channels with TEA and 4-AP increases the amplitude of somatic voltage signals and reduces attenuation into the axon (right). The vertical arrows denote the degree of steady-state attenuation in control and after K+ channel block. The traces in the two conditions are scaled differently to highlight the loss of axonal attenuation following K+ channel block.
(G) Summary of n = 15 experiments similar to (F), plotted as a function of distance of the axon bleb from the soma. The control data (black) are fit with a mono-exponential function yielding a length constant of 117 μm. The K+ channel block (red) significantly increases plateau propagation and doubles the functional length constant to 236 μm.
(H) Dual recording from soma and axon bleb (81 μm from soma) during depolarizing or hyperpolarizing steps injected at the soma (left). Of note is that the depolarizing step is comparatively more attenuated than the hyperpolarizing step. TEA/4-AP increases axonal propagation of the depolarizing step (right). The current injection was reduced in TEA/4-AP to maintain a depolarization of similar amplitude as in the control condition. The traces are from a different cell than (F).
(I) Mean voltage change at the soma and axon (x and y axes, respectively) during somatic current steps of varying polarity and amplitude. The data are from the same cell as (H). The shaded gray area and arrows signify the approximate range of depolarization observed during plateau potentials. The dotted lines are linear fits to the data during the hyperpolarizing steps in control and after K+ channel block (black and red, respectively). Under control conditions, somatic depolarizations >10 mV deviate significantly from the linear fit. Blocking K+ channels reduces this rectification.
(J) K+ channels limit axonal Ca2+ influx. Z stack of an example neuron filled with Alexa594 and the Ca2+ indicator OGB6F (200 μM) is shown. The roman numerals indicate the line scan locations at different points along the axon.
(K) Somatic voltage (upper trace) and fluorescence signals (lower traces) at axon locations denoted in (J). The black and red traces are from control conditions (in TTX) and after bath application of TEA/4-AP.
(L) Summary showing peak Ca2+ signals according to distance in control (black symbols) and after TEA/4-AP (red symbols). The data are fit with monoexponential functions. The data are pooled from n = 6 cells (71 and 88 line scan locations in control and TEA/4-AP, respectively).
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

6. Figure 5
Reliable and Selective Transmission of Na+ APs to Nerve Terminals during Complex Spikes
(A) 2-photon z stack of an example cell from which bouton Ca2+ transients were imaged using OGB6F. The arrow denotes the proximal axon. The dotted red box denotes the location of the imaged bouton.
(B) Top: high-magnification z stack of a bouton from the red rectangle in (A). Lower: the red line indicates the line scan location for fluorescence measurements. A complex spike (black) or temporally equivalent train of full-amplitude APs (gray) generates identical Ca2+ transients in the bouton.
(C) Summary for n = 10 boutons similar to (B). The red symbols are mean ± SEM.
(D) Lower traces: example traces of complex spikes or temporally equivalent full-amplitude AP trains in a presynaptic CA1 neuron (black and gray traces, respectively). The resulting average EPSP waveforms recorded in an interneuron are identical in the two conditions (upper traces).
(E) Amplitude of individual EPSPs evoked by each AP in a complex spike or full-amplitude AP train in n = 10 paired recordings. Of note is the identical release dynamics, indicating that presynaptic plateau waveforms do not enhance transmitter release from CA1 proximal boutons. A two-way repeated-measures ANOVA revealed a main effect of EPSP number (F(4,36) = 12.27, p < ). However, there was no effect of presynaptic waveform (F(1,9) = 1.027, p = 0.34) or any interaction between EPSP number and presynaptic waveform (F(4,36) = 1.089, p = 0.38). The error bars represent ±SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

7. Figure 6
Burst Firing and Distributed Network Activity Generate Distinct EPSP Dynamics in CA1 Downstream Targets
(A) Experimental setup. Whole cell recordings are made from pyramidal neurons in subiculum. Four stimulating electrodes are placed in the alveus region of CA1 (0.6–1.4 mm from the recorded neuron, minimum distance between stimulating electrodes: 0.1 mm).
(B) Upper traces: average unitary EPSPs evoked in a subiculum neuron after a single shock to each of four different alveus locations. Lower traces: the four locations are each stimulated once at 10 ms intervals (red trace). This protocol generates a compound EPSP resembling the waveform expected from the linear summation of the unitary EPSPs (black trace). The shorter half-width of the measured EPSP may be due to a non-linear recruitment of inhibition (of note is the dip below baseline in the red trace).
(C) Repetitive burst stimulation of each of the four pathways (four stimuli at 10 ms inter-stimulus intervals, red traces) generates greater EPSP summation than expected from the linear sum of the unitary events (black traces).
(D) Summary data for n = 7 cells plotting the ratio of measured/expected peak amplitudes for each EPSP in the train during distributed network activity (open triangles) or burst stimulation (black circles). A mixed effects model analysis revealed a significant effect of EPSP number for the burst inputs (F(3,94) = 16.58, p < ), but not for the distributed inputs (F(3,18) = 2.86, p = 0.07), along with a significant interaction between stimulus type and EPSP number (F(3,118) = 3.25, p = 0.02). Altogether, these data imply that burst inputs, but not distributed network patterns, summate supra-linearly in subiculum neurons. The error bars represent ±SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions