Non-additive effects of electrical stimulation of the dorsolateral prefrontal cortex and the vestibular system on muscle sympathetic nerve activity in humans.

Sinusoidal galvanic vestibular stimulation (sGVS) induces robust modulation of muscle sympathetic nerve activity (MSNA) alongside perceptions of side-to-side movement, sometimes with an accompanying feeling of nausea. We recently showed that transcranial alternating current stimulation (tACS) of the dorsolateral prefrontal cortex (dlPFC) also modulates MSNA, but does not generate any perceptions. Here, we tested the hypothesis that when the two stimuli are given concurrently, the modulation of MSNA would be additive. MSNA was recorded from 11 awake participants via a tungsten microelectrode inserted percutaneously into the right common peroneal nerve at the fibular head. Sinusoidal stimuli (± 2 mA, 0.08 Hz, 100 cycles) were applied in randomised order as follows: (i) tACS of the dlPFC at electroencephalogram (EEG) site F4 and referenced to the nasion; (ii) bilateral sGVS applied to the vestibular apparatuses via the mastoid processes; and (iii) tACS and sGVS together. Previously obtained data from 12 participants supplemented the data for stimulation protocols (i) and (ii). Cross-correlation analysis revealed that each stimulation protocol caused significant modulation of MSNA (modulation index (paired data): 35.2 ± 19.4% for sGVS; 27.8 ± 15.2% for tACS), but there were no additive effects when tACS and sGVS were delivered concurrently (32.1 ± 18.5%). This implies that the vestibulosympathetic reflexes are attenuated with concurrent dlPFC stimulation. These results suggest that the dlPFC is capable of blocking the processing of vestibular inputs through the brainstem and, hence, the generation of vestibulosympathetic reflexes.

The role of the dorsolateral prefrontal cortex in control of skin sympathetic nerve activity in humans.

The dorsolateral prefrontal cortex (dlPFC) is primarily involved in higher order executive functions, with there being evidence of lateralization. Brain imaging studies have revealed its link to the generation of skin sympathetic nerve activity (SSNA), which is elevated in states of emotional arousal or anxiety. However, no studies have directly explored dlPFC influences on SSNA. Transcranial alternating current stimulation (-2 to 2 mA, 0.08 Hz, 100 cycles) was applied between the left or right dlPFC and nasion via surface electrodes. Spontaneous bursts of SSNA were recorded from the common peroneal nerve via a tungsten microelectrode in 21 healthy participants. The modulation index was calculated for each stimulation paradigm by constructing cross-correlation histograms between SSNA and the sinusoidal stimulus. Stimulation of the dlPFC caused significant modulation of SSNA, but there was no significant difference in the median modulation index across sides. Stimulation also caused cyclic modulation of skin blood flow and sweat release. We have shown for the first time that stimulation of the dlPFC causes modulation of SSNA, also reflected in the effector-organ responses. This supports a role for the dlPFC in the control of SSNA, which likely contributes to the ability of emotions to bring about cutaneous vasoconstriction and sweat release.

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

PURPOSE: Sympathetic nerve activity towards muscle (MSNA) and skin (SSNA) regulates various physiological parameters. MSNA primarily functions in blood pressure and flow, while SSNA operates in thermoregulation. Physical and cognitive stressors have been shown to have effects on both types of sympathetic activity, but there are inconsistencies as to what these effects are. This article aims to address the discrepancies in the literature and compare MSNA and SSNA responses. METHODS: Microelectrode recordings were taken from the common peroneal nerve in 29 participants: MSNA (n = 21), SSNA (n = 16) and both MSNA and SSNA (n = 8). Participants were subjected to four different 2-min stressors: two physical (isometric handgrip task, cold pressor test) and two cognitive (mental arithmetic task, Stroop colour-word conflict test), the latter of which saw participants separated into responders and non-responders to the stressors. It was hypothesised that the physical stressors would have a greater effect on MSNA than SSNA, while the cognitive stressors would operate conversely. RESULTS: Peristimulus time histogram (PSTH) analysis showed the mental arithmetic task to significantly increase both MSNA and SSNA; the isometric handgrip task and cold pressor test to increase MSNA, but not SSNA; and Stroop test to have no significant effects on changing MSNA or SSNA from baseline. Additionally, stress responses did not differ between MSNA and SSNA in participants who had both sets of data recorded. CONCLUSIONS: This study has provided evidence to support the literature which claims cognitive stressors increase sympathetic activity, and provides much needed SSNA data in response to stressors.

Top-down control of vestibular inputs by the dorsolateral prefrontal cortex.

The vestibular apparatus provides spatial information on the position of the head in space and with respect to gravity. Low-frequency sinusoidal galvanic vestibular stimulation (sGVS), a means of selectively changing the firing of vestibular afferents, induces a frequency-dependent perception of sway and, in some individuals, induces nausea. Given that vestibular afferents project to the insular cortex-which forms part of the vestibular cortex-and that the insula receives inputs from the dorsolateral prefrontal cortex (dlPFC), we tested the hypothesis that electrical stimulation of the dlPFC can modulate vestibular inputs. Sinusoidal electrical stimulation (± 2 mA, 0.08 Hz, 100 cycles) was delivered via surface electrodes over (1) the mastoid processes alone (sGVS), (2) electroencephalogram (EEG) site F4 (right dlPFC) and the nasion or (3) to each site concurrently (sGVS + dlPFC) in 23 participants. The same stimulation protocol was used in a separate study to investigate EEG site F3 (left dlPFC) instead of F4 in 13 participants. During sGVS, all participants reported perceptions of sway and 13 participants also reported nausea, neither sensation of which occurred as a result of dlPFC stimulation. Interestingly, when sGVS and dlPFC stimulations were delivered concurrently, vestibular perceptions and sensations of nausea were almost completely abolished. We conclude that the dlPFC provides top-down control of vestibular inputs and further suggests that dlPFC stimulation may provide a novel means of controlling nausea.

Transient Thyroiditis Followed by Exacerbation of Hypothyroidism After Immune Checkpoint Inhibitor Therapy (Nivolumab and Ipilimumab) in a Patient With Pre-existing Autoimmune Hypothyroidism: A Case Report.

Treatment with immune checkpoint inhibitors has improved the prognosis of solid tumors. However, immune-related adverse events (IRAEs), including exacerbation of pre-existing autoimmune disease, are common and have become more frequent with combination therapy. The literature is scanty regarding reports of the use of combination immune checkpoint therapy in patients with pre-existing autoimmune hypothyroidism. We report a case of a man with a history of hypothyroidism, who developed transient thyroiditis, characterized by a thyrotoxic phase followed by a severe hypothyroid phase soon after receiving combination therapy (nivolumab and ipilimumab) for the treatment of a malignant pleural mesothelioma. He had been on a stable low dose of levothyroxine for 12 years prior to this episode. His levothyroxine requirement markedly increased soon after the episode of immune checkpoint inhibitor-induced thyroiditis. Immune checkpoint inhibitors can cause destructive thyroiditis followed by exacerbation of hypothyroidism in patients with pre-existing autoimmune hypothyroidism, such that patients end up on a higher dose of levothyroxine. This case will add to the growing literature regarding thyroid IRAEs associated with the use of immune checkpoint inhibitors in patients with pre-existing autoimmune thyroid disease.

Stimulation of the dorsolateral prefrontal cortex modulates muscle sympathetic nerve activity and blood pressure in humans.

Introduction: Muscle sympathetic nerve activity (MSNA) controls the diameter of arterioles in skeletalmuscle, contributing importantly to the beat-to-beat regulation of blood pressure (BP). Although brain imaging studies have shown that bursts of MSNA originate in the rostral ventrolateral medulla, other subcortical and cortical structures-including the dorsolateral prefrontal cortex (dlPFC)-contribute. Hypothesis: We tested the hypothesis that MSNA and BP could be modulated by stimulating the dlPFC. Method: dlPFC. In 22 individuals MSNA was recorded via microelectrodes inserted into the common peroneal nerve, together with continuous BP, electrocardiographic, and respiration.Stimulation of the right (n=22) or left dlPFC (n=10) was achieved using transcranial alternating current (tcACS; +2 to -2mA, 0.08 Hz,100 cycles), applied between the nasion and electrodes over the F3 or F4 EEG sites on the scalp. Results: Sinusoidal stimulation of either dlPFC caused cyclicmodulation of MSNA, BP and heart rate, and a significant increase in BP. Conclusion: We have shown, for the first time, that tcACS of the dlPFC in awake humans causes partial entrainment of MSNA, heart rate and BP, arguing for an important role of this higher-level cortical area in the control of cardiovascular function.

Functional analysis of cauliflower mosaic virus 35S promoter: re-evaluation of the role of subdomains B5, B4 and B2 in promoter activity.

The cauliflower mosaic virus 35S (35S) promoter is used extensively for transgene expression in plants. The promoter has been delineated into different subdomains based on deletion analysis and gain-of-function studies. However, cis-elements important for promoter activity have been identified only in the domains B1 (as-2 element), A1 (as-1 element) and minimal promoter (TATA box). No cis-elements have been described in subdomains B2-B5, although these are reported to be important for the overall activity of the 35S promoter. We have re-evaluated the contribution of three of these subdomains, namely B5, B4 and B2, to 35S promoter activity by developing several modified promoters. The analysis of beta-glucuronidase gene expression driven by the modified promoters in different tissues of primary transgenic tobacco lines, as well as in seedlings of the T(1) generation, revealed new facets about the functional organization of the 35S promoter. This study suggests that: (i) the 35S promoter truncated up to -301 functions in a similar manner to the -343 (full-length) 35S promoter; (ii) the Dof core and I-box core observed in the subdomain B4 are important for 35S promoter activity; and (iii) the subdomain B2 is essential for maintaining an appropriate distance between the proximal and distal regions of the 35S promoter. These observations will aid in the development of functional synthetic 35S promoters with decreased sequence homology. Such promoters can be used to drive multiple transgenes without evoking promoter homology-based gene silencing when attempting gene stacking.

Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping.

The cauliflower mosaic virus 35S (35S) promoter has been extensively used for the constitutive expression of transgenes in dicotyledonous plants. The repetitive use of the same promoter is known to induce transgene inactivation due to promoter homology. As a way to circumvent this problem, we tested two different strategies for the development of synthetic promoters that are functionally equivalent but have a minimum sequence homology. Such promoters can be generated by (a) introducing known cis-elements in a novel or synthetic stretch of DNA or (b) "domain swapping," wherein domains of one promoter can be replaced with functionally equivalent domains from other heterologous promoters. We evaluated the two strategies for promoter modifications using domain A (consisting of minimal promoter and subdomain A1) of the 35S promoter as a model. A set of modified 35S promoters were developed whose strength was compared with the 35S promoter per se using beta-glucuronidase as the reporter gene. Analysis of the expression of the reporter gene in transient assay system showed that domain swapping led to a significant fall in promoter activity. In contrast, promoters developed by placing cis-elements in a novel DNA context showed levels of expression comparable with that of the 35S. Two promoter constructs Mod2A1T and Mod3A1T were then designed by placing the core sequences of minimal promoter and subdomain A1 in divergent DNA sequences. Transgenics developed in tobacco (Nicotiana tabacum) with the two constructs and with 35S as control were used to assess the promoter activity in different tissues of primary transformants. Mod2A1T and Mod3A1T were found to be active in all of the tissues tested, at levels comparable with that of 35S. Further, the expression of the Mod2A1T promoter in the seedlings of the T1 generation was also similar to that of the 35S promoter. The present strategy opens up the possibility of creating a set of synthetic promoters with minimum sequence homology and with expression levels comparable with the wild-type prototype by modifying sequences present between cis-elements for transgene expression in plants.