A, confocal reconstruction of a CA3 pyramidal neurone filled with AlexaFluor 488 hydrazide. Image represents a maximum projection created from a 61 μm thick confocal image series. The asterisk marks the site of extracellular axon recording, measured in three dimensions at 641 μm from the soma. B, simultaneous somatic and axonal records from the imaged cell in A recorded in baseline conditions (2.5 mm[K⁺]_o). A 15 Hz action potential train was elicited by injecting a 90 pA depolarizing current at the soma (bottom trace). Extracellular action potential waveforms recorded at the axon were time locked to the somatic action potentials and were > 99% faithful in baseline conditions at all frequencies studied (up to ∼35 Hz). C, expanded display of the first action potential in the recording in B.

A, intracellular record of a train of action potentials elicited by a 1 s, 100 pA current injection at the soma from a resting potential of −70 mV. Trains at 10–30 Hz showed little accommodation at this stimulus strength and duration. B, plot of the first derivative of the somatic trace in A. C, extracellular ‘loose-seal’ recording at the soma of the cell shown in panels A and B. D–F, first action potentials from the train shown in A–C, respectively. Dotted vertical line indicates the peak of the somatic derivative (dV/dt). The extracellular waveforms peak at the same time as intracellular dV/dt, serving as a further indication that local capacitive currents (I_c=C_mdV/dt) dominate early portions of the extracellular waveform (see Fig. 4 for further discussion). G–I, intracellular somatic membrane potential, derivative (dV/dt), and extracellular axonal recordings in response to a 0.75 s, 200 pA current injection at the soma from a resting potential of −74 mV. Note the pronounced depression of maximum dV/dt later in the train. J–L, first action potentials from the trains shown in G–I. Despite the difference in signal/noise ratio, the polarity (positive) and kinetics are comparable to extracellular recordings at the soma. Vertical bar indicates the time of maximum somatic dV/dt. The peak of the axonal action potential occurs with a delay of 0.94 ms. Axonal latency varied with recording location (see Fig. 5C). Recording distance from the neurone/axon pair in G–L was measured in 3-dimensions to be 422 μm.

A, spiking-dependent depression of somatic and axonal signals during brief action potential trains. Values are normalized to the first action potential in the train. Somatic slope (filled bars) was depressed severely by action potential 10 (P < 0.01, n = 42 records from 36 neurones with mean frequency 23 ± 1 Hz). Axons from recordings immediately adjacent to the soma up to 800 μm away from the soma were pooled to yield estimates of axonal amplitude depression, which was much lower than somatic slope depression (open bars, P < 0.01, n = 36). B, the main plot (open symbols) represents axonal spike 10 amplitude depression during 15–30 Hz trains, plotted as a function of distance of the recording from the soma. The dotted line represents the degree of amplitude depression from 24 recordings performed > 150 μm from the soma. The inset (filled symbols) represents the same records after somatic slope depression was subtracted. Therefore, a value of zero indicates that somatic and axonal depression were exactly the same. The data were fitted by a single exponential rise to a maximum (continuous line, length constant: 102 μm). As indicated in Fig. 3, recordings < 30 μm from the soma may have been partially affected by local somatic signals. C, plot of axon–soma latency versus axonal recording distance. Negative values indicate the axon signal peaked before somatic dV/dt. Recordings up to 500 μm from the soma were fit with a bi-linear function of the form y=y_o+a(x−b) +c|x−b|. In this equation, b represents the distance corresponding to the nadir point (the average site of spike initiation). Antidromic conduction velocity can be calculated as 1/(c−a) and orthodromic conduction velocity 1/(c+a). Fit values for this data set reported an initiation site at 74.2 μm from the soma, an antidromic conduction velocity of 0.23 m s⁻¹, and orthodromic conduction velocity of 0.37 m s⁻¹.

A, somatic recording of an action potential train induced by a 50 pA step depolarization in 8 mm[K⁺]_o. Somatic resting membrane potential was −64 mV. B, first derivative plot of the action potential train in A. C, axonal action potential recording resulting from the action potential trains indicated in A and B. D, averaged responses of 10 cells at action potentials 1–10 from somata (•) and axons (▪) during action potential trains induced in 8 mm[K⁺]_o. As seen in baseline [K⁺]_o, somatic slope depression remained significantly greater than axonal amplitude depression (P < 0.01, n = 10). ○ and □, somatic slopes and axonal amplitudes (respectively) of action potentials 1–10 from these 10 neurones in 2.5 mm[K⁺]_o. E, instantaneous frequency response of somata and axons from action potentials 1 and 2 in 8 mm[K⁺]_o. Somatic slope (•) and axonal amplitude depression (▪) were slightly greater across all ISIs than controls (○ and □, respectively, P < 0.05, n = 10). Axonal spikes began to show frequency-dependent amplitude depression at ISIs < 25 ms in 8 mm[K⁺]_o, but did not depress more than 20%.

A, summary of somatic and axonal responses from 6 neurones that underwent PPs. Somatic slope depression in this data set was significantly reduced in 8 mm[K⁺]_o, both at spike 10 and during PPs (filled bars, P < 0.02 for both, n = 6). However, there was no additional slope depression on average during PPs than was seen by spike 10 of direct stimulation (P= 0.70, n = 6). In contrast, axonal amplitude was significantly reduced during PPs compared with spike 10 preceding the PP (open bars, P= 0.01, n = 6). B, effect of burst phenomena on axonal amplitude depression during PPs. Action potential bursts during PPs contained high-frequency spiking (> 20 Hz), and showed the most severe somatic slope and axonal amplitude reductions (see Fig. 8B, no. 4). Somatic action potentials occurring during PPs, but not involved in burst events (‘non-burst’ spikes) showed no significant change in slope depression (filled bars) with respect to the 10th spike before PP generation (P= 0.67). Despite this, axonal spike amplitude (open bars) during ‘non-burst’ events was significantly more depressed than the 10th spike preceding the PP (P < 0.01, n = 6). Somatic action potentials occurring during burst events (defined by instantaneous frequency > 20 Hz) showed significantly more slope depression and axonal amplitude depression than non-burst events during the PP (both P < 0.01). It should be noted that the strong axonal amplitude depression seen during bursts reduced these signals to the level of detection, and our measurements may represent an under-estimate of the actual axonal signal depression (Fig. 10). C, summary of the relationship between absolute somatic slope and axon amplitude before and during PPs. Somatic spikes with slopes below 90 mV ms⁻¹ during PPs (dark grey bars) were associated with more severe axonal amplitude depression than spikes with the same slopes in 2.5 mm[K⁺]_o (black bars) or 8 mm[K⁺]_o before PPs (light grey bars, P < 0.05, n = 6). At slope values above 90 mV ms⁻¹, axonal spike amplitudes in the 3 conditions were similar (P > 0.25, n = 6).

A, steady-state depolarization of the soma to a membrane potential between −45 and −65 mV (below spike threshold), followed by 1 s depolarizing steps (horizontal bar) resulted in PPs between 5 and 30 s in duration. B, from a membrane potential of ∼−70 mV, 25 s constant current injections caused plateau-like spiking. C, from a membrane potential of ∼−70 mV, brief (1–3 ms), high-intensity (1–3 nA) current pulses were delivered to the soma at 10 Hz. Although V_m did not recover to initial V_m between spikes, the steady-state V_m between spikes using pulse trains was well below that seen during the stimuli shown in A and B. All example traces were recorded from the same cell in 2.5 mm[K⁺]_o. Summary of somatic and axonal responses is shown in Fig. 12.

The primary image shows a CA3 neurone filled with biocytin. The neurone was visualized with streptavidin-conjugated Cy3 (red), and myelin was visualized with an antibody against myelin basic protein and secondary antibody conjugated to AlexaFluor 488 (green). Although occasional oligodendrocytes are present in the region (green labelling), we observed no colocalization of myelin staining with axons from the CA3 neurones studied (pseudocoloured pink to highlight, n = 22). The example is from a postnatal day 15 animal. The inset shows myelin basic protein staining in the corpus callosum from the same slice at the same depth of focus, exposure, and gain settings as the primary panel. Both images represent a confocal projection 50 μm thick from the surface of the slice.

A, extracellular recordings taken at short distances from the soma display negative local potentials (inset) and decay exponentially with a length constant of 12.5 μm. The exponential fit to this curve is shown by the dashed line. The horizontal dotted line represents detection threshold. Inset: when the extracellular suction electrode was brought into contact with the somatic membrane, the ‘loose seal’ configuration was achieved as seen in Fig. 3A–F (pipette resistance: 6.8 MΩ). A positive signal is measured, similar in polarity and kinetics to axonal signals. The polarity and kinetics indicate this initial signal results from capacitive current flow across the membrane. Ionic currents are likely to contribute to later portions of the waveform (Jack et al. 1975). When displaced small distances from the somatic membrane and suction applied to maintain relative pipette resistance (3.5–4.2 MΩ), local potentials reverse direction, indicating a flow of positive charges (source current) out of the extracellular electrode during somatic spiking. The pipette thus contributes to the local source currents within 25–30 μm of the soma. Traces represent spike-triggered averages of 8 isolated somatic spikes. Calibration bar represents 2 ms and 100 μV. B, extracellular potential when the extracellular electrode was placed ∼5 μm from a visualized axon, 567 μm from the soma. Suction was applied (pipette resistance changed from ∼3 to ∼6 MΩ) as during axonal recording. No extracellular waveform was observed. Traces represent spike-triggered averages of 8 individual spikes. C, when the axon was brought into the lumen of the extracellular electrode following suction (pipette resistance 6.5 MΩ), a local positive potential was recorded at specific latencies from the somatic spike (see Fig. 5C).

A, soma current clamp plots from a CA3 neurone with spontaneous variations in interspike interval (ISI) between action potentials 1 and 2 induced by DC injection. At the smallest ISIs, somatic action potential amplitude was reduced and recovered with increasing ISI. B, first derivative plot of the trace shown in A. Whereas somatic amplitude was reduced by < 20% at the smallest ISIs, these amplitude reductions were accompanied by slope reduction of > 50%. C, extracellular axonal recording from the cell in A. Axonal action potential amplitude was not depressed, even at the smallest spontaneous ISIs. D, averaged ISI-dependent changes from 279 spontaneous variations in ISI from 18 neurones. Responses were binned with 5 ms intervals with the exception of the last 2 ISI bins (60–70 ms and 70–105 ms, respectively). Soma amplitude (grey circles) and slope (•) recovered with increasing ISI, whereas axonal amplitude (□) showed no substantial depression at any of these intervals.

A, CA3 somata often responded to stimulation in elevated [K⁺]_o with 10–50 s PPs. During these PPs, the soma fired action potentials at 5–50 Hz, with average spike frequency only 13 ± 2 Hz (n = 6). B–D, somatic and axonal action potentials on a smaller time scale. Recordings 1–5 represent the segments indicated by the numbered bars below the trace in A. B, axonal action potentials decreased in amplitude during PPs (nos 2–5), but not before (no. 1). Asterisks mark peak times of somatically detected spikes. C, soma derivative plot shows severe dV/dt depression during PPs, especially during burst events (nos 2 and 4). D, somatic action potential amplitude from current clamp traces shows much less severe depression in absolute amplitude during PPs, even during burst events.

A, raw extracellular spikes from a paired somatic–axonal recording during a PP. Axonal traces were aligned based on the peak of somatic dV/dt of spikes during a PP in 8 mm[K⁺]_o. Condition labels appear to the left of each trace. ‘Initial’ spike represents the first spike in response to the 1 s current injection. Individual ‘non-burst’ and ‘burst’ detection failures are indistinguishable from noise. Scale bar: horizontal 1 ms, vertical 100 μV. B, somatic dV/dt-triggered averages of 18 axonal responses in each of the categories to the left in A except the initial spike, which is reproduced for reference. Spike-triggered averaging for ‘non-burst’ spikes revealed detectable, but strongly diminished, axonal waveforms. In contrast, spike-triggered averaging from ‘burst’ spikes revealed no detectable waveform (compare with averaged noise, bottom trace), indicating propagation failure. C, scatter plot of individual amplitudes for burst (filled circles) and non-burst (open circles) events during a single PP. We observed no failures before spike 60 in this PP (5.1 s from the initial spike). The horizontal dotted line represents detection threshold. As seen in the raw traces, burst and non-burst detection failures are not easily distinguishable from one another on an individual basis. D, average amplitudes from successful detections and failures during the PP. Non-burst detection failures, while having an average amplitude below detection threshold (horizontal dotted line), had a significantly larger amplitude than burst failures. Asterisk indicates P < 0.01. Results are representative of 2 of 4 plateau cells with sufficient numbers of failure events from bursts and non-bursts for spike-triggered averaging. The remaining two cells had no discernible waveform in spike-aligned averages from either non-burst failures or burst failures, indicating that outright failure is also possible during non-burst spikes in some cells.

A, binned record from an example neurone during 3 PPs induced in 2.5 mm[K⁺]_o utilizing the protocol shown in Fig. 11A. The somatic membrane potential was depolarized using a steady-state current injection to −58 mV followed by a 1 s DC pulse to induce a brief 20 Hz action potential train. This spiking was followed by PPs lasting 22 ± 2 s. Soma slope (•) decreased strongly during the 1 s pulse (preceding the arrow), followed by a partial recovery associated with the decrease in spike frequency from 20 to 8 Hz. Axonal spike amplitude (□) became progressively depressed throughout this period, and did not recover along with somatic slope during the transition to PP spiking. The horizontal dotted line indicates axonal detection threshold. B, binned record from the same neurone in A in response to a 25 s sustained current injection from a resting potential of −72 mV (see Fig. 11B). The nature of the depolarizing pulse did not induce high-frequency action potential spiking initially, but the average frequency was similar to the other method (9 Hz compared to 8 Hz during the PP in A). The decline in somatic slope (•) and axonal amplitude (□) was similar to the other method. Grey diamonds indicate the axonal spike amplitude depression caused by 10 Hz pulse trains as shown in Fig. 11C. Note this depression was substantially smaller than either of the other conditions. C, summary of responses of 5 neurones exposed to the spiking paradigms represented in Figs 8 and 11. Axonal spike amplitude depression seen using the methods in Fig. 11A and B were statistically indistinguishable from 8 mm[K⁺]_o-induced PPs (filled bars: somatic slope reduction; open bars: axonal action potential depression). Ten-hertz, 200 pulse trains in these 5 neurones (rightward-most open bar) reported similar spike amplitude reduction at spikes 175–200 to reductions seen at the 10th spike before PP induction. This value was statistically less affected than all sustained depolarization conditions (P < 0.05 for all, n = 5).