The 5-HT system is involved in the regulation of cognitive functions such as learning and short- and long-term memory 2,13−15 and thus has become a pharmacological and genetic target for the treatment of memory disorders 5,16. Serotonin signaling is mediated by 14 receptor subtypes with different functional and transduction properties. The 5-HT1A subtype is of particular interest since it is one of the main mediators of the action of 5-HT and plays an important regulatory role in the 5-HT system 5,17−19. Perez-Garcia G et al. speculated that the mRNA expression of 5-HT1A receptor in important brain regions may be used as a specific neuromarker of explicit memory and implicit memory 20. Elucidating the transcription and regulation of 5-HT1A receptor will help to determine its mode of action in the nervous system.
The transcription of the rat 5-HT1A receptor gene is mainly initiated by the sequence from − 967 bp upstream to the ATG segment of the start codon 11. The site of rs198585630, which was identified in our previous study, is located in the promoter region of the 5-HT1A receptor gene. No functional studies on this site have been reported yet. The premise for studying the functional significance of rs198585630 as a SNP in the promoter region is that regulation of 5-HT1A receptor expression has physiological and pathological significance. Our previous study indicated that variations in 5-HT1A receptor expression are involved in microwave exposure-induced cognitive deficits. rs198585630, which is located in the promoter region, has the potential to regulate the expression of 5-HT1A receptor. Therefore, in vivo and in vitro experiments were conducted to study the function of rs198585630 and its association with individual differences in cognitive alterations after microwave exposure.
An in vitro study indicated that the transcriptional activity of 967-bp 5-HT1A receptor fragments containing the − 215 C allele was higher than that of 967-bp 5-HT1A receptor fragments containing the T allele. The SNP rs198585630 may be located at the TFBS, and the − 215 T > C polymorphism affected transcription activity. Moreover, promoters containing the C allele were more sensitive to alterations in transcriptional activity after microwave exposure than those containing the T allele.
Based on the in vitro study of rs198585630 function, in vivo studies were carried out to explore the associations between rs198585630 and alterations in 5-HT1A receptor induced by microwave exposure. The mRNA and protein expression levels of 5-HT1A receptor in the rats’ hippocampi of the TC and CC genotypes increased significantly after exposure, which was basically consistent with the results of in vitro experiments. rs198585630 significantly affected the expression of 5-HT1A receptor. The rs198585630 C allele is related to higher expression of 5-HT1A receptor and more sensitive to microwave exposure. Studies in rat and mouse models have provided evidence that performance in the MWM test is highly sensitive to changes in 5-HT1A receptor function 20–22. Considering the important role of 5-HT1A receptor in cognitive function, we tested the spatial learning and memory of rats of different genotypes and found that among rats in the sham exposure groups, the spatial learning ability of rats of the TT genotype was lower than that of rats of the TC and CC genotypes. The spatial learning ability of the TT genotype exposed to microwaves was enhanced compared to that of control of the same genotypes, whereas spatial memory was not significantly altered after microwave exposure. The spatial learning and memory of the TC and CC genotypes were both significantly reduced after exposure.
An increase in 5-HT1A receptor density is related to a decline in cognitive function 23–25, and 5-HT1A receptor expressed on hippocampal postsynaptic neurons has a negative effect on explicit memory function 18,26. Stimulation of 5-HT1A receptors generally results in learning impairments by interfering with memory-encoding mechanisms, while antagonists of 5-HT1A receptors facilitate certain types of memory by enhancing hippocampal/cortical cholinergic and/or glutamatergic neurotransmission 20,27,28. Similarly, we observed that compared with the rs198585630 T allele, the rs198585630 C allele was related to higher transcriptional activity of the 5-HT1A receptor promoter and increased mRNA and protein expression of 5-HT1A receptor. Rats carrying the rs198585630 C allele were more susceptible to the spatial learning and memory deficits induced by microwave exposure.
The 5-HIAA/5-HT ratio reflects the relative metabolic rate of 5-HT, which can be used to evaluate serotonergic system activity 29–31. After microwave exposure, the 5-HIAA/5-HT ratio in rs198585630 TT rats was significantly decreased, reflecting decreased catabolism and increased excitability of 5-HT neurons. Considering the performance of the rats in the MWM test (increased spatial learningand no significantly change in memory), the decrease in the 5-HIAA/5-HT ratio may represent a mechanism compensating for decreased expression of 5-HT1A receptor and leading to a return of the 5-HT system to the physiological range to maintain effective neurotransmission.
Alterations in EEGs can be seen in pathology and cognitive disorders, as these conditions involve cognitive deficits that are closely related to inhibition of EEG activity 32–34. The monoamine-acetylcholine balance hypothesis is a theory related to neurophysiological markers on EEG, and an increased delta frequency band reflects an increase in the effects of inhibitory monoamine receptor subtypes such as 5-HT1A receptor 35. At a low dosage, a 5-HT1A receptor agonist enhances EEG power in delta rage 36. The EEG results showed that after 30 mW/cm2 microwave exposure, the delta band relative powers of rats in the TC and CC genotype groups were higher than that of rats in the TT genotype group, which was consistent with the alterations in 5-HT1A receptor expression after microwave exposure described previously.
In summary, the rs198585630 site in the rat 5-HT1A receptor promoter region is a functional site that regulates the transcription of 5-HT1A receptor. The transcriptional activity of the 5-HT1A receptor promoter containing the − 215 C allele is higher than that of the 5-HT1A receptor promoter containing the T allele. The transcriptional activity of the 5-HT1A receptor promoter was stimulated by 30 mW/cm2 microwave exposure, and the 5-HT1A receptor rs198585630 C allele was more sensitive to microwave exposure, showing stronger transcriptional activation. Rats with the rs198585630 C allele presented higher mRNA and protein expression of 5-HT1A receptor and were more susceptible to 30 mW/cm2 microwave exposure, as indicated by cognitive deficits and brain electrical activity inhibition, than those with the rs198585630 T allele. These findings suggest that the SNP rs198585630 of the 5-HT1A receptor is an important target for further research exploring the mechanisms of hypersensitivity to microwave exposure.