Use-dependent regulation of the axonal action potential in parvalbumin-expressing interneurons

Reviewer #1 (Public review):

Summary:

This paper examines whether action potentials (APs) reliably propagate to the distal axon in neocortical parvalbumin-expressing interneurons (PV-Ins) during prolonged high-frequency activity, as occurring during epileptiform activity. The authors use dual soma and axon-attached patch-clamp recordings from mouse and human PV-INs and show that axon AP amplitude declines when the firing frequency exceeds ~200 Hz and fails during seizure-like bursts. Finally, they show that elevation of external K+ to 10 mM also reduces AP amplitude. Taken together, these data strongly suggest that the reduction in transmitter release observed during intense PV-INs activity or during seizure-like events is mainly mediated by the reduction in the presynaptic AP amplitude in PV-INs.

Strengths:

This paper is very interesting, well-written and technically impressive. It provides new and important results. The paper will have a great impact in the field of both axon physiology and epilepsy.

Weaknesses:

I did not find any significant weakness in the methods, data analysis and results.

Reviewer #2 (Public review):

Summary:

The authors demonstrate a frequency-dependent progressive failure of action potential propagation through the axonal arbors in fast-spiking interneurons

Strengths:

The experimental protocols are technically challenging, but the data is of very high quality, and the presentation and writing are very clear.

I congratulate the authors on submitting a really excellent study demonstrating an activity-dependent alteration in the efficacy of axonal propagation of action potentials in fast-spiking interneurons. It is a well-designed project involving technically challenging experiments, and yet the data is of very high quality, the results are compelling, and the presentation is clear.

Weaknesses:

I have some minor suggestions and comments, including those below, but I hope and expect that these could be performed quickly and without difficulty.

Two of the most interesting figures were consigned to the supplementary information, and I would recommend that they are "upgraded" to be in the main document. The two figures are Figure 1 - Figure Supplement 2, showing the inverse correlation of the AP size with recording distance and branch; and Figure 6 - Figure Supplement 1, showing the postsynaptic effect. My rationale for saying this is that I feel that both add useful biological information to the narrative.

I was glad to see that "realistic" firing patterns were used, because I recall an old modelling paper from Mainen and Sejnowski (https://pubmed.ncbi.nlm.nih.gov/7770778/) that is highly relevant to this paper and should be referenced. However, I would like to suggest one further bit of analysis of the data presented in Figure 4, because I think it will support the main story. In Figure 4, the ostensible conclusion is that there is relative preservation of spike amplitude for this natural firing pattern, but that is almost certainly because the average firing rate is substantially below the level where spike amplitude suppression was seen in Figures 2 & 3. Instead, I recommend analysing for each consecutive spike pair, the ratio of the heights of the two spikes with respect to the interspike interval. Viz
t2 - t1 versus spike 2 amplitude / spike 1 amplitude

The data may be a little noisy, but given the very large number of spike pairs, I would expect to see the suppression effect to be fully evident, and that can feed directly into the model.
I think the author's intuition that dissipation of ionic gradients is a key factor is correct, so I was pleased that Na+ was not ignored in the discussion (the results section only talked about K+).

Perhaps the fact that Na gradients may also be depleted could be mentioned in the results section, too. In the discussion, perhaps the authors could mention two other details: that this "fatigue" may reflect ATP depletion, and progressive failure of the Na-K-ATPase in the axons. That could be examined in a follow-up study (I certainly am not suggesting a raft of experiments for this study), but it could be mentioned in the discussion. And second, that the ionic depletion may be greater within the confines of the cell-attached pipette tip, which is why the branching pattern/distance data (F1FS2), the Ca imaging data and the post-synaptic effects (F6FS1) are such important additional supporting data, because together they indicate that the effect is along the whole axon.

Regarding the rise in [K+]o, it would be worth mentioning the fact that this will be greatly exacerbated by the postsynaptic effects of high-frequency PV activity, because the consequent Cl loading of the postsynaptic cell is subsequently cleared by coupling to K+ extrusion. A good reference for this is http://www.ncbi.nlm.nih.gov/pubmed/20211979; a recent review (https://pubmed.ncbi.nlm.nih.gov/39637123/), which argues that this may even be the dominant source of raised [K+]o in the immediate preictal period, larger even than that exiting cells through the Hodgkin-Huxley mechanism.

The referencing needs some attention. In some instances, the citations either do not really illustrate preceding statements or are simply the wrong citation.

Reviewer #3 (Public review):

Summary:

This is an interesting paper which asks a compelling and translationally relevant question: since the firing rate of GABAergic PV+ interneurons (which powerfully control pyramidal cell excitability) increases prior to and during seizures, why doesn't this increase in inhibition do more to prevent seizure propagation? The authors hypothesize that increased PV+ spiking might lead to spike propagation failures in the axon.

To test this hypothesis, the authors conduct paired electrophysiological recordings from PV+ neurons in acute barrel cortex slices of mice and from a handful of human neurosurgical samples. They use patch clamp recordings to measure the membrane potential of PV+ neurons at the soma, while simultaneously measuring spike propagation with a recording electrode in the axon of the same neuron.

After a variety of elegant experiments and modeling, the authors conclude that extracellular K+ accumulation around the axon during high-frequency firing might be causing propagation failures.

Strengths:

Overall, the paper is nicely written, the experiments are technically challenging, and the figures are, for the most par,t well laid out. The topic will be of broad interest for the neuroscience field, given the relevance of PV+ interneurons to cortical circuit function, plasticity across development, and disease.

Weaknesses:

In addition to the strengths here, I feel the need to highlight a few weaknesses which, if rectified, could improve the work.

(1) The key hypothesis in this paper is that extended periods of somatic spiking lead to progressive decreases in the axonal AP amplitude, which eventually lead to failures, potentially (but not necessarily) at branchpoints. Two comments here.

It would be helpful for the authors to show us examples of the axonal spike waveforms at a faster time base (along with the somatic recording) so that we can really understand what's happening to the spike in the axon.

Their data are also compatible with failures of spike initiation at the AIS. Could the authors show us the first derivative of somatic voltage for successes and failures, and maybe show us some phase plots of Vm vs dV/dt for the failures, successes, and attenuated spikes? Effectively, what I'm asking is whether the changes they see in the distal axon are downstream of the initiation zone. It's very possible that extended spiking is simply depolarizing the AIS and inactivating Na+ channels there. In which case, the authors should be able to pull this out in a phase plot.

(2) There's no baseline period for their calcium fluorescence signals, which is necessary to compare their "signal" magnitude to frame-by-frame variance of dG/R. Could the authors correct this issue in Figure 6B?

(3) Some of their stats are a bit unorthodox. Why are they doing two separate Wilcoxon tests in 6D and 6E? Why not throw all that into a one-way ANOVA model followed by appropriate post hoc tests?

(4) Why don't the authors observe washout of their effect after high K+ application? This concerns me that their high K+ application is having secondary and long-lasting effects on PV excitability, which mimic (but are not necessarily identical) to their hypothesized mechanism of axonal failures.