Neuromodulation of key proteins within the PG is possible via invasive electrical stimulation of the sympathetic pathway . Modulation of salivary proteins has also been shown through both invasive and non-invasive electrical stimulation [20–22]. However, ours is the most comprehensive study to date investigating both intracranial (PG) and extracranial (salivary and lacrimal glands) changes in gene expression in response to non-invasive electrical stimulation at different dermatomes and frequencies.
Our research shows stimulation at the C2 dermatome can modulate expression of genes encoding melatonin machinery with frequency-specific effects. These effects might be mediated via a transcriptional regulatory mechanism that encodes extracellular signals as bursts of nuclear localization of a transcription factor responsible for activating specific GOI . In this manner, higher frequency stimulation is potentially necessary to stimulate both the pineal and submandibular glands’ innervation pathways, and lower frequency stimulation favors that of the parotid gland. Indeed, low frequency (20 Hz) electrical stimulation can enhance parotid protein output in sheep  and high frequency (50 Hz) stimulation can increase saliva secretion from the submandibular gland in rats . This frequency preference between the glands could be attributed to differing gene expression profiles that have been noted between the parotid and the submandibular glands [26, 27], which is supported by our data showing very low expression of GOI in the parotid gland and moderate expression in the submandibular gland. Such frequency-specific effects between structures are not unusual and have been documented extensively in previous literature surrounding neuromodulation.
We observed a change in Hiomt expression levels but saw no change in Aanat. Whilst Aanat upregulation is driven by adrenergic input from the post-ganglionic sympathetic fibres, HIOMT activity can be significantly increased in vitro via acute administration of neuropeptide Y (NPY). Moreover, no change in HIOMT activity occurs following administration of the β-adrenergic receptor agonist, isoproterenol, nor the α1-adrenergic receptor agonist, phenylephrine . This suggests that, unlike AANAT, rapid changes in pineal HIOMT activity are regulated via a noradrenergic-independent mechanism. The PG possesses NPY-ergic fibres in a variety of mammals such as the rat [29–31], Syrian hamster , guinea pig , cow , cat , monkey , and human . Moreover, NPY is co-localized with NE in perivascular, sympathetic nerve terminals [38, 39]. The SCG provides perivascular innervation to the pineal as perivascular nerve terminals disappear from the gland following bilateral SCG-ectomy [40, 41]. Release of NPY from such terminals shows a preference for receiving stimulation at a higher frequency compared to lower frequency . NPY is known to inhibit pineal melatonin release via inhibiting the stimulatory effect of NE on pinealocytes [43–45]. In this context, 80 Hz stimulation at the C2 dermatome in rats is potentially exerting its effect through the sympathetic innervation route of the PG via a potential NPY-ergic mechanism, causing an increase in Hiomt expression.
A previous study by Brownstein and Heller found a decrease in HIOMT levels following invasive stimulation of the preganglionic sympathetic fibres . Methodological differences likely account for why we instead found an increase in Hiomt expression. Firstly, they stimulated at a frequency of 10 Hz, whereas we observed no change following 10 Hz and a significant upregulation with 80 Hz. This indicates higher frequencies can induce an stimulatory effect on Hiomt, whereas lower frequencies cannot, further emphasizing frequency-specific effects on pineal neuromodulation. Secondly, Brownstein and Heller found a decline in HIOMT levels with stimulation periods greater than two hours and did not sacrifice animals immediately following stimulation cessation, whereas we stimulated for two hours and opted for immediate sacrifice. Waiting one hour prior to sacrificing stimulated animals causes AANAT levels to decline  indicating that without immediate sacrifice and extraction of pineals following stimulation, any increase in AANAT levels might not be observable. Moreover, two hours of stimulation optimally upregulates AANAT levels, with a decline observed following three hours of stimulation . Based on these observations, longer stimulation times offer a further explanation for why Brownstein and Heller observed a decrease in HIOMT levels.
Modulatory effects of C2 dermatome stimulation on the salivary glands are likely due to them receiving sympathetic innervation from the SCG which are also associated with innervation of the C2 dermatome, as previously described. The lack of any modulatory effects via T1 dermatome stimulation may be due to the absence of such anatomical connections between the T1 level of the spinal cord and these glands. Previous invasive stimulation studies all utilized either pre- or post-ganglionic fibres or the cervical sympathetic trunks  and, to the best of our knowledge, no other studies have attempted to non-invasively stimulate the SCG and ICG separately via their corresponding dermatomes.
Retinoic acid is a potent transcriptional and translational regulator , involved in signalling and regulation within the PG . Rarβ is a noted tumor suppressor gene  and encodes for a retinoic acid receptor. Its expression is either low or silenced in various cancerous cells [51–57] and its increase in expression in these cells increases their sensitivity to chemotherapeutic agents, making it an ideal chemosensitisation target . As 80 Hz stimulation at the C2 dermatome was able to significantly upregulate Rarβ expression within the PG, utilization of this effect to may help sensitize PG tumours to chemotherapeutic agents and improve the likelihood of positive treatment outcomes.
With regards to our upregulation of Rorβ in the parotid gland, this effect was observed following both 10 and 80 Hz stimulation indicating that this effect is not frequency-specific, but dermatome-specific. Retinoic acid-related orphan receptors (RORs) are a subfamily of nuclear hormone receptors for which a high-affinity, endogenous ligand has not been identified. RORs are not only involved in the modulation of circadian rhythms but also have important roles in the progression of certain cancers as both a tumour suppressor agent  and a tumour-promoting agent . Research into Rorβ functioning and activity in this regard is still in its infancy with no clear identification of its role in oncogenesis and cancer progression. Therefore, the extent of the clinical significance for the increase in expression in the parotid gland in this study cannot yet be identified.
Our 10 Hz stimulation increased Mt3 expression in the submandibular gland. Whilst named as a third melatonin receptor, MT3 has a greater affinity for the melatonin precursor NAS . NAS has greater antioxidant and free-radical scavenging abilities than even melatonin [62–64] and this antioxidant effect might be mediated via the MT3 receptor [65–68]. There exists evidence for melatonin-independent roles for NAS in vivo as it can activate the tropomyosin receptor kinase B receptor which is involved in mediating the effects of brain-derived neurotrophic factor, whereas melatonin cannot . Further, as much as 15% of melatonin synthesized is converted back to NAS [70, 71] and both NAS and melatonin are secreted from the PG . Since it has previously been shown that melatonin is synthesized and likely secreted from the submandibular glands [73, 74], they may also secrete NAS. If this is true, then perhaps our 10 Hz stimulation at the C2 dermatome may promote an antioxidant effect via increased NAS signalling. Further, as brain-derived neurotrophic factor decreases with age [75, 76] and is also a potent regulator of plasticity [77, 78], it plausible to suggest that low frequency stimulation of the C2 dermatome could have potential as an adjunct for dementia prevention .
The overall low expression of most of our GOI within the parotid gland suggests that the synthesis, regulation, and signalling of melatonin within this gland is extremely low during the night. Moreover, despite melatonin being present in human tears  and the presence of AANAT, HIOMT with the lacrimal glands of Syrian hamsters , our experiments indicate that the expression of both Aanat and Hiomt within the lacrimal gland are also extremely low. This suggests that the rat lacrimal gland is not an extrapineal site of melatonin synthesis during the night, thus explaining a lack of any modulatory effect observed here.
Due to the tiny size of the PG, utilization of the whole gland for RNA extraction was necessary to collect enough total RNA for downstream analysis by qPCR. This meant there was not enough tissue available for assaying protein levels. As gene expression does not necessarily directly equate to protein levels, with correlation between the two estimated to be as little as 40% [82, 83], our study is limited to only making inferences regarding modulatory changes in gene expression. Therefore, for example, whilst we may have observed an increase in Hiomt expression, this might not necessarily equate to an increase in HIOMT activity.
Further, as our experiments were conducted in rats, any clinical implications drawn from our findings are merely speculative as there is no guarantee that similar results would be achieved with the same stimulation in humans. Therefore, future work is necessary to determine whether the results can be replicated in humans, and whether the stimulation protocols are able to elicit changes in both gene expression, corresponding protein levels, and what effect this may have on subsequent melatonin levels.
We have shown that non-invasive electrical stimulation of the C2 dermatome can modulate gene expression within the pineal, submandibular, and parotid glands with frequency-specific effects. Also, to the best of our knowledge, we have demonstrated the expression levels of Mt3, Rorβ, Tph1 within the submandibular gland; Hiomt, Clock, Bmal1, Mt3, Rarβ, Rorβ, Rev-erbα, Tph1, Cry1, within the parotid gland, and Aanat, Hiomt, Gapdh, Mt3, Rorβ, Rev-erba, and Tph1 within the lacrimal gland of the rat for the first time. As Aanat expression did not change with stimulation, our findings offer further evidence of a non-adrenergic innervation pathway to the PG through which Hiomt expression is regulated. This may occur through a NPY-ergic mechanism. These results have potential clinical applications in the sensitization of pineal tumour cells to chemotherapeutic agents via Rarβ upregulation. Future directions for this research should investigate the mechanism behind such upregulation, if changes in gene expression correspond to changes in protein levels within the same glands, and whether clinical translation of these findings is possible.