Diet therapy as a treatment strategy for epilepsy has a long history. For instance, KD, as a well-known low-carb, high-fat diet, is widely used in the treatment of epilepsy, autism spectrum disorders and Alzheimer's disease [35, 36]. However, to date, there is no effective diet intervention for SUDEP prevention. Alteration of dietary TRP is often used as a noninvasive method to manipulate the TRP levels of the body, thereby affecting 5-HT neurotransmission in the CNS . HTD has been applied in the treatment of fatty liver disease , diabetes , Alzheimer's disease  and so on, which indicates that HTD is safe and feasible as an adjunctive therapy. Our study is the first to demonstrate that HTD is an effective diet intervention in preventing SUDEP in DBA/1 mice.
In this study, we found that an HTD significantly increased 5-HT levels in the telencephalon. Previous studies showed that SSRIs significantly increased the 5-HT content in the frontal cortex of rats . Extensive synaptic connections were found between the cortex of the telencephalon and 5-HT neurons in the dorsal raphe of the midbrain sublattice . In addition, some scholars found that telencephalon was also involved in the arousal mechanism of consciousness disorders . Therefore, we hypothesized that the increase in telencephalon 5-HT levels may affect S-IRA occurrence in DBA/1 mice through the neural network between the telencephalon and midbrain. We also found that the HTD significantly increased 5-HT levels in the midbrain. Our previous study showed that the occurrence of S-IRA was significantly inhibited by selectively activating 5-HT neurons in the midbrain through optogenetic technology in transgenic DBA/1 mice . We speculated that the HTD reduced S-IRA in this SUDEP model due to the elevation of the 5-HT concentration in the midbrain. Interestingly, the 5-HT level was not significantly altered in the pons and medulla of DBA/1 mice after the HTD, which differs partially from the results of Zhan’s study in which multiunit recordings showed decreased firing of neuron populations both in the medullary and midbrain raphe, and single-unit recordings of serotonergic neurons revealed consistently decreased firing in the medullary raphe but a mixture of increased and decreased firing in the midbrain raphe during the ictal and postictal periods of an established Sprague Dawley (SD) rat seizure model . We conjectured that the inconsistency may be due to strain differences and seizure triggering methods between DBA/1 mice and SD rats. As the literature stated, the midbrain raphe may be more likely involved in the mechanism of unconsciousness, the medullary raphe may be more involved in cardiorespiratory dysfunction during and after epileptic seizures in SD rats , while the midbrain may be more critical in S-IRA in this SUDEP model. In the future, the specific roles of these two nuclei and whether selectively activated 5-HT neurons in the medulla are associated with a reduced incidence of SUDEP should be studied further.
We found that the species abundance and diversity of the gut microbial community of animals in the ND group was less than that of animals in the HTD group. There was an obvious difference in the intestinal flora composition between the HTD and ND groups, and the gut microbiota relative abundance of the HTD-treated mice was dominated by Proteobacteria and Actinobacteria. The mechanisms underlying bacterial-induced 5-HT signalling are not well understood. Studies proved that some Proteobacteria and Actinobacteria species were closely related to the increase in short-chain fatty acids (SCFAs) , which were reported to be capable of promoting 5-HT production in peripheral blood . Since we did not test the SCFA differences in the HTD and ND groups, we did not know if the HTD reduced the S-IRA rate by affecting the gut microbiota and then elevating SCFAs and eventually peripheral and central 5-HT. In addition, some studies found that local stimulation of gut metabolites mediated by certain intestinal flora can regulate brain activity through the autonomic nervous system , and the stimulation of peripheral vagal nerves could modulate the regulation of central 5-HT . Other bacterial species have also been reported to be capable of modulating 5-HT metabolism. For example, the administration of lipopolysaccharide, a cytoderm component of gram-negative bacteria, significantly increased the production of 5-HT in the prefrontal cortex, striatum and midbrain of animals [50, 51], possibly via the modulation of TPH activity . In addition, some bacterial metabolites, such as acetic acid (an SCFA), can regulate the expression of serotonin receptors in the gut and brain, as well as change behaviours in animals . Generally, the exact mechanism by which the gut microbiota mediates the changes in 5-HT levels in the CNS through the "gut-brain axis" is relatively complicated and still needs further exploration.
A previous study found that the levels of 5-HT and 5-HIAA in the hippocampus of male GF animals were significantly higher than those in the corresponding controls . It is therefore natural to speculate that antibiotics would further elevate brain 5-HT levels in DBA/1 mice, which would accordingly enhance the inhibitory effect of HTD on SUDEP. Moreover, antibiotics did not exert synergistic effects with the HTD for S-IRA suppression. This finding should not be ascribed to the difference in the intestinal environment between GF animals and antibiotic-treated animals because first, in our study, there were also elevated levels of 5-HT and 5-HIAA in the midbrain of the antibiotic treatment group compared with the control group (as shown in Supplementary Fig. 1), and second, intestinal environment testing showed that antibiotics inhibited most of the intestinal flora of DBA/1 mice (as shown in Supplementary Fig. 2), which was consistent with previous reports [53, 30]. These observations somehow equated antibiotic-treated animals to GF animals. It is more likely that the production of central 5-HT in DBA/1 mice reached saturation after administration of the HTD, thus, the combination of the HTD and antibiotics could not further increase the concentration of 5-HT in the CNS (see Supplementary Fig. 3).
Probiotics or prebiotics intervention is closely related to peripheral and central 5-HT metabolism in animals. For instance, Lactobacillus plantarum PS128 (PS128) significantly augmented striatal 5-HT levels in GF mice . Oral Lactobacillus reuteri 3 (L. reuteri 3) notably elevated 5-HT levels in the periphery and colon of depressed mice . At present, it is believed that the possible mechanism by which probiotics or prebiotics affect central 5-HT is related to their effect on the reconstruction of TRP metabolism and 5-HT transformation , but the exact mechanism is unclear. In our study, the HTD combined with probiotics was not superior to the HTD alone in SUDEP prevention. This combination did not significantly change the metabolism of TRP and 5-HT in plasma or the CNS (as shown in Supplementary Fig. 4). We deduced that, on the one hand, supplementation with probiotics may not produce a superimposition effect on the HTD-mediated 5-HT increase in the CNS. On the other hand, the HTD combined with probiotics may alter the regulation of central 5-HT by directly changing the intestinal flora spectrum or indirectly affecting certain metabolites of the "gut-brain axis". Another possible explanation is that Lactobacillus, Proteobacteria and Actinobacteria modulated 5-HT metabolism through the same pathway in the "gut-brain axis", but this was not revealed by our current study and still needs further research.
Taking into consideration the important role of central 5-HT synthesis in SUDEP, it is meaningful to detect 5-HT deficiency in patients with epilepsy, which is helpful in differentiating those who are at high risk for SUDEP. Recent studies found that PET/SPECT could monitor alterations of the 5-HT receptor/5-HT transporter in associated brain regions by serotonergic probes  or regional blood flow  and had been applied in neuropsychiatric and neurodegenerative disorders [58, 59]. Therefore, screening patients with serotonin-targeted PET/SPECT is a promising prospect for the prevention and treatment of SUDEP in the future.
In conclusion, our research was the first to demonstrate that an HTD significantly reduced the incidence of S-IRA in DBA/1 mice, possibly mediated by gut microbiota, which affected the synthesis and metabolism of 5-HT, primarily in the CNS. However, the specific molecular mechanism remains to be further clarified. Our findings may open another window for the pathogenesis of SUDEP, and an HTD is expected to be a promising candidate for the prevention of SUDEP in clinical practice, especially for patients with central serotonin deficiency.