Squalamine reverses age-associated changes of firing patterns of myenteric sensory neurons and vagal fibres.

Vagus nerve signaling is a key component of the gut-brain axis and regulates diverse physiological processes that decline with age. Gut to brain vagus firing patterns are regulated by myenteric intrinsic primary afferent neuron (IPAN) to vagus neurotransmission. It remains unclear how IPANs or the afferent vagus age functionally. Here we identified a distinct ageing code in gut to brain neurotransmission defined by consistent differences in firing rates, burst durations, interburst and intraburst firing intervals of IPANs and the vagus, when comparing young and aged neurons. The aminosterol squalamine changed aged neurons firing patterns to a young phenotype. In contrast to young neurons, sertraline failed to increase firing rates in the aged vagus whereas squalamine was effective. These results may have implications for improved treatments involving pharmacological and electrical stimulation of the vagus for age-related mood and other disorders. For example, oral squalamine might be substituted for or added to sertraline for the aged.

Identification of SSRI-evoked antidepressant sensory signals by decoding vagus nerve activity.

The vagus nerve relays mood-altering signals originating in the gut lumen to the brain. In mice, an intact vagus is required to mediate the behavioural effects of both intraluminally applied selective serotonin reuptake inhibitors and a strain of Lactobacillus with antidepressant-like activity. Similarly, the prodepressant effect of lipopolysaccharide is vagus nerve dependent. Single vagal fibres are broadly tuned to respond by excitation to both anti- and prodepressant agents, but it remains unclear how neural responses encode behaviour-specific information. Here we demonstrate using ex vivo experiments that for single vagal fibres within the mesenteric neurovascular bundle supplying the mouse small intestine, a unique neural firing pattern code is common to both chemical and bacterial vagus-dependent antidepressant luminal stimuli. This code is qualitatively and statistically discernible from that evoked by lipopolysaccharide, a non-vagus-dependent antidepressant or control non-antidepressant Lactobacillus strain and are not affected by sex status. We found that all vagus dependent antidepressants evoked a decrease in mean spike interval, increase in spike burst duration, decrease in gap duration between bursts and increase in intra-burst spike intervals. Our results offer a novel neuronal electrical perspective as one explanation for mechanisms of action of gut-derived vagal dependent antidepressants. We expect that our ex vivo individual vagal fibre recording model will improve the design and operation of new, extant electroceutical vagal stimulation devices currently used to treat major depression. Furthermore, use of this vagal antidepressant code should provide a valuable screening tool for novel potential oral antidepressant candidates in preclinical animal models.

Squalamine Restores the Function of the Enteric Nervous System in Mouse Models of Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative disorder thought to be caused by accumulation of α-synuclein (α-syn) within the brain, autonomic nerves, and the enteric nervous system (ENS). Involvement of the ENS in PD often precedes the onset of the classic motor signs of PD by many years at a time when severe constipation represents a major morbidity. Studies conducted in vitro and in vivo, have shown that squalamine, a zwitterionic amphipathic aminosterol, originally isolated from the liver of the dogfish shark, effectively displaces membrane-bound α-syn. OBJECTIVE: Here we explore the electrophysiological effect of squalamine on the gastrointestinal (GI) tract of mouse models of PD engineered to express the highly aggregating A53T human α-syn mutant. METHODS: GI motility and in vivo response to oral squalamine in PD model mice and controls were assessed using an in vitro tissue motility protocol and via fecal pellet output. Vagal afferent response to squalamine was measured using extracellular mesenteric nerve recordings from the jejunum. Whole cell patch clamp was performed to measure response to squalamine in the myenteric plexus. RESULTS: Squalamine effectively restores disordered colonic motility in vivo and within minutes of local application to the bowel. We show that topical squalamine exposure to intrinsic primary afferent neurons (IPANs) of the ENS rapidly restores excitability. CONCLUSION: These observations may help to explain how squalamine may promote gut propulsive activity through local effects on IPANs in the ENS, and further support its possible utility in the treatment of constipation in patients with PD.

Microvesicles from Lactobacillus reuteri (DSM-17938) completely reproduce modulation of gut motility by bacteria in mice.

Microvesicles are small lipid, bilayer structures (20-400 nm in diameter) secreted by bacteria, fungi, archaea and parasites involved in inter-bacterial communication and host-pathogen interactions. Lactobacillus reuteri DSM-17938 (DSM) has been shown to have clinical efficacy in the treatment of infantile colic, diarrhea and constipation. We have shown previously that luminal administration to the mouse gut promotes reduction of jejunal motility but increases that in the colon. The production of microvesicles by DSM has been characterized, but the effect of these microvesicles on gastrointestinal motility has yet to be evaluated. To investigate a potential mechanism for the effects of DSM on the intestine, the bacteria and its products have here been tested for changes in velocity, frequency, and amplitude of contractions in intact segments of jejunum and colon excised from mice. The effect of the parent bacteria (DSM) was compared to the conditioned media in which it was grown, and the microvesicles it produced. The media used to culture the bacteria (broth) was tested as a negative control and the conditioned medium was tested after the microvesicles had been removed. DSM, conditioned medium, and the microvesicles all produced comparable effects in both the jejunum and the colon. The treatments individually decreased the velocity and frequency of propagating contractile cluster contractions in the jejunum and increased them in the colon to a similar degree. The broth control had little effect in both tissues. Removal of the microvesicles from the conditioned medium almost completely eradicated their effect on motility in both tissues. These results show that the microvesicles from DSM alone can completely reproduce the effects of the whole bacteria on gut motility. Furthermore, they suggest a new approach to the formulation of orally active bacterial therapeutics and offer a novel way to begin to identify the active bacterial components.

Colonic Motility and Jejunal Vagal Afferent Firing Rates Are Decreased in Aged Adult Male Mice and Can Be Restored by an Aminosterol.

There is a general decline in gastrointestinal function in old age including decreased intestinal motility, sensory signaling, and afferent sensitivity. There is also increased prevalence of significant constipation in aged populations. We hypothesized this may be linked to reduced colonic motility and alterations in vagal-gut-brain sensory signaling. Using in vitro preparations from young (3 months) and old (18-24 months) male CD1 mice we report functional age-related differences in colonic motility and jejunal mesenteric afferent firing. Furthermore, we tested the effect of the aminosterol squalamine on colonic motility and jejunal vagal firing rate. Old mice had significantly reduced velocity of colonic migrating motor complexes (MMC) by 27% compared to young mice (p = 0.0161). Intraluminal squalamine increased colonic MMC velocity by 31% in old mice (p = 0.0150), which also had significantly reduced mesenteric afferent single-unit firing rates from the jejunum by 51% (p < 0.0001). The jejunal vagal afferent firing rate was reduced in aged mice by 62% (p = 0.0004). While the time to peak response to squalamine was longer in old mice compared to young mice (18.82 ± 1.37 min vs. 12.95 ± 0.99 min; p = 0.0182), it significantly increased vagal afferent firing rate by 36 and 56% in young and old mice, respectively (p = 0.0006, p = 0.0013). Our results show for the first time that the jejunal vagal afferent firing rate is reduced in aged-mice. They also suggest that there is translational potential for the therapeutic use of squalamine in the treatment of age-related constipation and dysmotility.