Differential control of sympathetic outflow to muscle and skin during physical and cognitive stressors

Our isometric handgrip and cold pressor tasks increased MSNA from baseline to a statistically significant extent by the last 50 R-wave time period. This was not an unexpected result, as a wealth of literature exists verifying the efficacy of these tasks in positively modulating MSNA [4, 23, 28‐30] and our own cumulative burst amplitude analysis corroborated these results. Moreover, the cold pressor task saw a sustained increase from baseline during the 50 R-wave recovery period, which was not reflected in the handgrip task. As such, these results suggest that the isometric handgrip task is capable of producing a robust, but more quickly resolving, increase in MSNA—the immediate relaxation reflecting the immediate cessation of central command. The increase in MSNA seen during the cold pressor test developed slowly but continued into the immediate recovery period. These PSTH results are incongruent with the results obtained from the standard metrics (Table 1), however. The cold pressor test did not see an increase in burst frequency or incidence, which is somewhat unexpected given the widely cited effects of this stressor on MSNA [23, 30]. One possible explanation for this comes back to the properties of PSTH analysis and the nature of the cold pressor test. During this stressor, participants had a tendency to tense up as a result of pain and discomfort—often leading to extraneous signals in the microneurographic recording. This inevitably led to messier data for use in standard metric reporting, while PSTH analysis filtered out this signal disturbance and, hence, led to results more in line with the current literature.

Mental arithmetic, just as with the isometric handgrip and cold pressor tasks, increased MSNA from baseline to a statistically significant extent by the last 50 R-wave time period, but was the only stressor to produce a significant increase in spike count within the first 50 R-waves, indicating a more rapid influence of this stressor on sympathetic outflow to the muscle vascular bed. The efficacy of the mental arithmetic task was further exemplified when the data was separated into responder and non-responder groups, given the evidence that mental stressors can induce positive or, indeed, negative influences on MSNA on an individual basis [31, 32]. An additional difference was identified between the two time points during the stressor. Results such as these are supported by Wang et al.’s study [33] in which they reported that various cardiovascular responses had rapid increases during the first 150 s of mental arithmetic before plateauing. As our own mental arithmetic protocol was conducted for only 120 s, it is reasonable to assume subjects were still within the rapid increase period. While the consistency of the task at increasing MSNA is contentious, our results and experimental design differ from two of the more commonly cited papers displaying its inhibitory properties. Firstly, Matsukawa et al. [15] used the tibial nerve at the popliteal fossa for their microneurographic recordings. As they themselves state, another group [11] demonstrated discrepancies in levels of MSNA during mental arithmetic based on the recorded nerve. Furthermore, Delius et al. [14] had a sample size of only four for their mental arithmetic task and did not account for what we now know to be inter-individual variability in cognitive stressor response (i.e. responders and non-responders). Other studies which suggest that mental arithmetic does not increase MSNA [16‐20] also do not separate responders and non-responders, resulting in an averaged response in which the increase in MSNA in the responders and decrease in the non-responders essentially cancel each other out. With this being said, our own data on the standard metrics (i.e. burst frequency, burst incidence and cumulative burst amplitude) aligns with these studies—that is, our mental arithmetic protocol saw a decrease in MSNA from baseline. As such, sympathoinhibition associated with mental arithmetic is no doubt real. Additionally, we identified a lack of a response during the Stroop colour–word conflict test in the PSTH pooled data group. Thus, participants were subsequently separated into responders and non-responders. Through this analysis it was found that the last 50 R-wave time period significantly differed from the recovery period, but still no significant changes were found from baseline. In a possible explanation for this, Callister et al. [34] proposed that MSNA fluctuations during mental stress tests are directly correlated to the perceived level of stress during the test. As our MSNA participants reported a relatively low average stress rating of 3.1 ± 1.7 arbitrary units out of 10 (obtained from 20 of the 21 participants), this would appear to be expected. The six responders had a similar stress rating of 3.2 ± 1.7. However, the accuracy of the conclusion offered by Callister et al. [34] is yet to be further proven, with studies such as the one produced by Carter et al. [16] contradicting the original hypothesis.

The results obtained from the SSNA data set are somewhat less supported by the current body of literature, though SSNA has been far less studied than MSNA. The cold pressor test did not induce any significant increase in spike counts, as one might expect from prior work [6]. Moreover, there were no differences in direct comparison of their efficacy in altering SSNA from baseline. More surprisingly, however, handgrip also did not increase SSNA from baseline to a significant extent. This result is incongruent with prior works showing isometric handgrip tasks to have profound effects on SSNA [28, 35, 36]. Each of these studies used a relatively similar handgrip and nerve recording protocol to our own—the primary discrepancy lies in the method chosen to analyse SSNA data: using the integrated nerve signal or using the PSTH approach we used herein. Analysis conducted on the cold pressor and handgrip manoeuvres using the RMS-processed nerve signal, however, did indicate increases in SSNA during the stressors when compared to their baseline and recovery periods.

The lack of significant differences from baseline found in the physical stressors using PSTH analysis also carried over to the cognitive stressors. As SSNA is tied to an individual’s emotions [37, 38], this may be expected of the Stroop test (with the low average stress rating of 3.5 ± 2.0 arbitrary units out of 10), but mental arithmetic is known to have profound effects on SSNA in both healthy and diseased states [12, 39, 40]. Once the cognitive stressors had been separated into responders and non-responders, though, mental arithmetic showed an increase in SSNA by the last 50 R-wave time period. Therefore, it can be concluded that the non-responders dampened the results in the pooled data, leading to the lack of a significant increase. Granted, Muller et al. [12] identified increases in SSNA immediately upon commencement of the stressor, but given the lack of reports on mental arithmetic and SSNA of healthy controls with respect to time, further investigation is needed to corroborate these results. Furthermore, the Stroop test still did not reveal significant changes from baseline post-data separation. What it did reveal was a change in SSNA between the last 50 R-waves of the stressor and the recovery period. Alongside the fact that the first 50 R-waves of the stressor no longer had a statistically significant difference with the recovery period, it may be likely that the small sample size of six diminished the power of the results. This would therefore lead to the lack of significant differences between baseline and the stress condition. The one difference found between the stressor and non-stressor time points is consistent with what one would expect from SSNA (i.e. stress increases SSNA), as discussed by Carter and Goldstein [18], so it may be reasonably assumed that a larger sample of cognitive-stress responders would have led to expected data.

No differences of statistical significance were found between any two corresponding time points in the eight participants from which we were able to gather both MSNA and SSNA data. It has been previously shown that sympathetic nerve activity does not vary to a great extent within an individual over the course of months to even years [6] in the absence of disease. From this knowledge it may be safe to assume our data were not skewed by the time interval of approximately 1–4 months between visits. Our data suggests that each of the stressors has the same level of impact on both MSNA and SSNA within individuals. That is, an individual will react in the same manner regarding muscle and skin response when subjected to stress.

One of the primary limitations of this study involved the inability to conduct each stress procedure on every participant, reducing the sample sizes for the experiment. Overall, this was not a great issue, with sample sizes reflecting and, in some cases, exceeding prior work; however, this became a more pronounced limitation in the comparison of MSNA and SSNA within participants. Here, the sample size was only eight and further reduced for three of the four stressors. As such, this served to undermine the accuracy of the results for this section of the analysis—especially since the cognitive stressors could not be separated into responders and non-responders. Additionally, with regards to the sample, experiments were conducted on a young, healthy cohort. While this does mean the results presented are representative of the healthy population, they do not reflect stress responses in the elderly or in disease states, which may warrant further investigation. It may also be worth noting that the menstrual cycle of the female participants was not accounted for. There are differing results in the literature as to its effects on sympathetic nerve activity, with some studies reporting no changes during rest and cognitive stressors, and other studies showing profound increases in resting MSNA depending on the stage of the cycle [31, 41]. Nevertheless, there is a possibility it may have impacted our results. Furthermore, our experiments were conducted in a thermoneutral environment (room temperature 22 °C), yet past studies have gone a step further and implemented the use of a customised tube-lined suit to maintain neutral skin temperature [12, 36]. Experimenting in such a way would perhaps account for some of the discrepancies in our SSNA results.