Effects of nicotine compared to placebo gum on sensitivity to pain and mediating effects of peak alpha frequency

Reviewer #1 (Public Review):

Summary:
In this study, Millard and colleagues investigated if the analgesic effect of nicotine on pain sensitivity, assessed with two pain models, is mediated by Peak Alpha Frequency (PAF) recorded with resting state EEG. The authors found indeed that nicotine (4 mg, gum) reduced pain ratings during phasic heat pain but not cuff pressor algometry compared to placebo conditions. Nicotine also increased PAF (globally). However, mediation analysis revealed that the reduction in pain ratings elicited by the phasic heat pain after taking nicotine was not mediated by the changes in PAF. Also, the authors only partially replicated the correlation between PAF and pain sensitivity at baseline (before nicotine treatment). At the group-level no correlation was found, but an exploratory analysis showed that the negative correlation (lower PAF, higher pain sensitivity) was present in males but not in females. The authors discuss the lack of correlation.
In general, the study is rigorous, methodology is sound and the paper is well-written. Results are compelling and sufficiently discussed.

Strengths:
Strengths of this study are the pre-registration, proper sample size calculation, and data analysis. But also the presence of the analgesic effect of nicotine and the change in PAF.

Weaknesses:
It would even be more convincing if they had manipulated PAF directly.

Reviewer #2 (Public Review):

Summary: The study by Millard et al. investigates the effect of nicotine on alpha peak frequency and pain in a very elaborate experimental design. According to the statistical analysis, the authors found a factor-corrected significant effect for prolonged heat pain but not for alpha peak frequency in response to the nicotine treatment.

Strengths: I very much like the study design and that the authors followed their research line by aiming to provide a complete picture of the pain-related cortical impact of alpha peak frequency. This is very important work, even in the absence of any statistical significance. I also appreciate the preregistration of the study and the well-written and balanced introduction. However, it is important to give access to the preregistration beforehand.

Weaknesses: The weakness of the study revolves around three aspects:

(1) I am not entirely convinced that the authors' analysis strategy provides a sufficient signal-to-noise ratio to estimate the peak alpha frequency in each participant reliably. A source separation (ICA or similar) would have been better suited than electrode ROIs to extract the alpha signal. By using a source separation approach, different sources of alpha (mu, occipital alpha, laterality) could be disentangled.

(2) Also, there's a hint in the literature (reference 49 in the manuscript) that the nicotine treatment may not work as intended. Instead, the authors' decision to use nicotine to modulate the peak alpha frequency and pain relied on other, not suitable work on chronic pain and permanent smokers. In the present study, the authors use nicotine treatment and transient painful stimulation on non-smokers.

In my view, the discussion could be more critical for some aspects and the authors speculate towards directions their findings can not provide any evidence. Speculations are indeed very important to generate new ideas but should be restricted to the context of the study (experimental pain, acute interventions). The unfortunate decision to use nicotine severely hampered the authors' aim of the study.

Impact: The impact of the study could be to show what has not worked to answer the research questions of the authors. The authors claim that their approach could be used to define a biomarker of pain. This is highly desirable but requires refined methods and, in order to make the tool really applicable, more accurate approaches at subject level.

Reviewer #3 (Public Review):

In this manuscript, Millard et al. investigate the effects of nicotine on pain sensitivity and peak alpha frequency (PAF) in resting state EEG. To this end, they ran a pre-registered, randomized, double-blind, placebo-controlled experiment involving 62 healthy adults who received either 4 mg nicotine gum (n=29) or placebo (n=33). Prolonged heat and pressure were used as pain models. Resting state EEG and pain intensity (assessed with a visual analog scale) were measured before and after the intervention. Additionally, several covariates (sex at birth, depression and anxiety symptoms, stress, sleep quality, among others) were recorded. Data was analyzed using ANCOVA-equivalent two-wave latent change score models, as well as repeated measures analysis of variance. Results do not show *experimentally relevant* changes of PAF or pain intensity scores for either of the prolonged pain models due to nicotine intake.

The main strengths of the manuscript are its solid conceptual framework and the thorough experimental design. The researchers make a good case in the introduction and discussion for the need to further investigate the association of PAF and pain sensitivity. Furthermore, they proceed to carefully describe every aspect of the experiment in great detail, which is excellent for reproducibility purposes. Finally, they analyze the data from almost every possible angle and provide an extensive report of their results.
The main weakness of the manuscript is the interpretation of these results. Even though some of the differences are statistically significant (e.g., global PAF, pain intensity ratings during heat pain), these differences are far from being experimentally or clinically relevant. The effect sizes observed are not sufficiently large to consider that pain sensitivity was modulated by the nicotine intake, which puts into question all the answers to the research questions posed in the study.