Evidence suggests that the gut bacteria are a source of sleep-inducing signals11,12, and we hypothesized that SCFAs may serve as such signal. Our major finding is that orally or intraportally administered tributyrin and butyrate, respectively, robustly increases NREMS in rats and mice. These observations are consistent with prior reports that intravenous injection of butyrate induces slow, high-amplitude EEG waves and behavioral signs of sleep in rabbits34 or EEG-defined NREMS in cats35.

Butyrate is a four-carbon SCFA, produced by the microbiota in mice and rats. It is the product of the anaerobic fermentation of non-digestible carbohydrates by gut bacteria and also a component of dairy products, such as butter, milk and cheese36. Tributyrin is an ester, composed of three butyric acid molecules and glycerol. It is considered a pro-drug to deliver biologically active butyrate as lipases in the host organism hydrolyze it resulting in the release of butyrate37. SCFAs, including butyrate, are readily absorbed from the intestines into the portal circulation and directly reach the liver29. Plasma levels of butyric acid in the portal circulation after oral administration of tributyrin are higher and more prolonged without detectable toxicity in mice and rats as compared to administration of butyrate itself38. To mimic the effects of intestinally produced butyrate, we administered tributyrin orally to mice. This treatment elicited an almost 50% increase in NREMS in the first 4 h supporting the notion that butyrate from the intestinal tract may potentially serve as a sleep-inducing signal molecule. NREMS increased at the expense of both REMS and wakefulness. REMS suppression may be due to the mutual inhibitory interaction between NREMS- and REMS-promoting mechanisms39 or it could be a NREMS-independent effect of tributyrin. The treatments were administered at dark onset. Since the latency to increased sleep is very short and the duration of the sleep increases did not exceed 8 hours, the effects of butyrate on sleep were manifested predominantly during the dark, active, phase.

Sleep-inducing doses of butyrate also elicited a 0.4–1.2 °C drop in body temperature. Since naturally occurring NREMS is associated by decreased energy expenditure and body temperature (reviewed in40), it is possible that the slight hypothermic response is simply the thermic manifestation of enhanced NREMS after butyrate treatment. It has been proposed that a drop in core body temperature prompts sleepiness41, thus an alternative interpretation is also possible, i.e., an initial drop in body temperature in response to butyrate may invoke the sleep responses.

Baseline sleep recordings were performed on the day before the butyrate treatment. In thoroughly-habituated rats and mice, such as our experimental animals, sleep and body temperature are remarkably stable across two successive days. This is evidenced, for example, by the experiments where mice received ip injection of saline on day 1 and butyrate on the following day (Fig. 3). Thus, it is highly unlikely that the observed sleep-promoting effects of intraportally or orally administer butyrate are confounded by order of the treatments.

There is a steep concentration gradient between the high portal levels of butyrate and very low butyrate concentration in the systemic circulation29,30,42,43, and orally administered tributyrin increases portal, but not systemic, levels of butyrate44. These observations indicate that the liver removes almost all butyrate from the portal blood31. Thus, the most likely target for butyrate to induce sleep is the hepatoportal system. To investigate this possibility, we injected butyrate directly into the portal vein in rats. Intra-portal butyrate treatment greatly reduced NREMS latency and increased the time spent in NREMS. To investigate, if a potential butyrate overflow from the liver into the systemic circulation could be responsible for the sleep effects, we injected the same amount of butyrate systemically. Systemic administration of butyrate did not have any effect on sleep in rats. Similarly, none of the systemically-administered doses of butyrate had any effect on sleep-wake activity in mice. These findings indicate that the sleep effects of orally or intraportally administered butyrate are not due to the actions of butyrate that possibly escaped the hepatic sink. We conclude that butyrate acts on the liver and/or the portal vein to promote NREMS. There is prior evidence that the liver is involved in peripheral sleep signaling, since local warming of the liver increases NREMS8, and depletion of liver Kupffer cells impairs recovery sleep responses after sleep loss and sleep in a cold environment45.

Hepatoportal sensors have been described for several gut-derived molecules, e.g., glucose, cholecystokinin, and amino acids46,47,48. They are located in the wall of the portal vein and the liver and have been implicated in the regulation of glucose and energy homeostasis (reviewed in49). There is also evidence for hepatoportal SCFA sensors. Butyrate receptors are present in the hepatoportal region. Butyrate signals through the receptors FFAR2, FFAR3 and GPR109A, all of which is expressed in the liver32,33,50, thus may serve as hepatic sensors. FFAR3 is also expressed by the portal vein wall in close proximity to neuronal markers51. The activation of these receptors affects brain circuits as evidenced by the observation that the effects of dietary SCFAs on the activity of the nucleus tractus solitarius and parabrachial nucleus are abolished by the selective sensory denervation of the periportal area51. The sensory innervation of the hepatoportal region is provided by the vagus and spinal afferents, both of which have been implicated in sleep signaling52,53,54,55. Butyrate directly activates vagal afferents56 and the effects of butyrate on feeding is suppressed by hepatic vagotomy57.

Several bacterial-derived sleep-inducing molecules, such as lipopolysaccharide and fragments of peptidoglycans, have been described before (reviewed in58). All these molecules are components of the bacterial cell wall, they are released from disintegrating bacteria or from bacteria during cell division. They have pronounced inflammatory actions via the stimulation of the production of pro-inflammatory cytokines (reviewed in59). Sleep responses to systemic bacterial infection are linked to these pro-inflammatory processes. The properties of butyrate are, however, fundamentally different. Butyrate is produced by live bacteria in the intestines, and it has strong anti-inflammatory properties. It suppresses colonic and liver inflammation and lipopolysaccharide-induced production of pro-inflammatory cytokines and NF-κB activation26,60,61,62,63. This indicates that not only systemic pro-inflammatory signals related to bacterial infections, but also bacterial-derived anti-inflammatory signals from the intestinal tract have the potential to modulate sleep.