Single-axon action potentials in the rat hippocampal cortex

Abstract

Whether all action potentials propagate faithfully throughout axon arbors in the mammalian CNS has long been debated, and remains an important issue because many synapses occur far from the soma along extremely thin, unmyelinated, varicosity-laden branches of axon arbors. We detected unitary action potentials along individual axon branches of adult hippocampal CA3 pyramidal cells using extracellular electrodes, and analysed their conduction across long distances (mean, 2.1 mm) at 22 and 37 degrees C. Axons nearly always transmitted low-frequency impulses. At higher frequencies, most axons also transmitted impulses with striking fidelity. However, at paired-pulse frequencies in the hundreds of kilohertz range, axons exhibited variability: refractory periods ranged from 2.5 to 10 ms at 37 degrees C and from 5 to 40 ms at 22 degrees C. Although the basis for the refractory period variability could not be determined, these limits overlap with CA3 spike frequencies observed in vivo, raising the possibility that some axonal branches act as filters for the higher-order spikes in bursts, in contrast to the observed first-spike reliability. These results extend the observations of propagation reliability to a much longer distance and higher frequency domain than previously reported, and suggest a high safety factor for action potential propagation along thin, varicose axons.

Figure 1. Morphological features of DiI-labelled CA3-to-CA1 axons, and the drawing up by negative pressure of a flexible loop of axon into a recording pipette

A, distal branches of CA3 axons travelling in area CA1 are often branched on a scale of hundreds of micrometres. A crystal of DiI was deposited in area CA1 radiatum of an acute slice in an interface chamber and incubated for several hours to label CA3-to-CA1 axons. The panel shows a tracing of the trajectory and branching pattern of a DiI-labelled axon. The number of branches formed by axons varied. Over distances of 1 cm or so (the greatest extent of labelling in these acute experiments), axons often branched several times, as seen in this example. Occasional axons had no identifiable branches over such distances. B, a confocal microscope image of a DiI-labelled axon showing that at higher magnification and resolution, CA3-to-CA1 axons are highly varicose. The straightness of this axonal branch on this scale is also typical for these axons. C, the same pressure parameters used for physiological single-axon recording caused individual DiI-labelled axons to be gently pulled into the recording pipette, as visualised in these examples using epifluorescence/differential interference contrast imaging. Varicosities along the axons are evident, but the axonal shaft segments between varicosities are often fainter due to their small diameter and the rapid bleaching of the dye that occurs during the procedure. The example on the left shows an axon that appears to enter and leave the pipette in an Ω form, whereas the one on the right may have been broken at one end. Some unlabelled axons presumably also entered the pipette together with the labelled one. These pictures demonstrate that axons could be sucked into pipettes with tip openings of 5 and 15 μm (left and right panel, respectively), but such visualisation was not used during the experiments in which electrical recordings were made.

Figure 2. Single-axon action potentials

A, action potentials (asterisks) were recorded extracellularly from an axon in the CA1 stratum radiatum. B, spikes from A are shown aligned, and on a faster time scale. C, activation of single-axon action potentials (arrow) by minimal stimulation. Suprathreshold stimuli of varying intensities evoked spikes that varied little in waveform and latency (arrow, upper traces). Subthreshold stimuli did not evoke responses (lower traces). D, stimulus-response relationship of a single axon, showing that spike amplitude (peak-to-peak) was constant above the activation threshold (arrowhead) of μ75 μs. Five failures (arrows) out of 154 stimuli above threshold were not related to the stimulus strength. E, the upper trace shows activation of both the unit shown in C and D (asterisk) and a second, earlier unit (arrow) detected with slightly stronger stimuli (average of 30 traces). The response to stronger stimulation (lower trace) shows that single units had latencies within the time window of the population response. F, the latency of the unit from C–E was highly stable on a time scale of minutes. G, eight units from different experiments, normalised with respect to their peak-to-peak amplitude. stim, point at which the stimulus was given.

Figure 3. The proportion of failures was low

A, the total number of failures and successes, normalised, at high and low temperature. B, a cumulative plot of the success proportion for eight units at 22 °C and 13 units at 37 °C.

Figure 4. Analysis of the shortest response interval for one axon at two temperatures

A, action potentials (asterisks) were evoked by paired stimuli (same intensity, 27 ms interval). B, first- and second-spike amplitudes as a function of stimulus intensity. C, responses to paired suprathreshold stimuli with interstimulus intervals of 1–40 ms. The top traces are superimposed responses to the first stimulus in each pair. The bottom traces are responses to the second stimulus in each pair, offset vertically for display according to the pair's interstimulus intervals (randomly ordered during the experiment). For the shortest intervals, no second responses occurred (marked ‘fail’). Traces at these short intervals did contain first responses, which appeared because of the short interstimulus intervals. D, ratios of second/first response amplitudes, as a function of the interstimulus interval.

Figure 5. Average results at high temperature and low temperature

A, the minimum (threshold) intervals for eliciting a second response. B, the amplitude and latency of the second response, relative to the first.

Figure 6. Comparison of compound and unitary action potential refractory periods

A, upper trace, typical compound action potentials (asterisks) from a population of CA3-to-CA1 axons, elicited with two identical stimuli separated by 7 ms. Lower traces, responses at two temperatures evoked by paired pulses separated by intervals of 1–40 ms (presented in random order). Second-stimulus artefacts have been removed. B, peak-to-peak amplitude of the second response, normalised to the first, as a function of the interstimulus interval. Lines represent double-exponential fits to the data. C, the fast exponential functions measured from populations of axons (continuous lines) closely matched the distributions of refractory periods measured from single axons (circles).