Astrocytes ACLYmate to chronic neuroinflammation.

Astrocytes are essential cells of the mammalian central nervous system (CNS), with key roles in development, homeostasis, and disease. Lee and colleagues recently showed that astrocytes can develop epigenetic memory, which enhances proinflammatory responses to subsequent stimulation, potentially driving sustained neurological disease pathology, such as in multiple sclerosis (MS).

An electroencephalographic examination of the autonomous sensory meridian response (ASMR).

Autonomous sensory meridian response (ASMR) is a perceptual phenomenon characterized by pleasurable tingling sensations in the head and neck, as well as pleasurable feelings of relaxation, that reliably arise while attending to a specific triggering stimulus (e.g., whispering or tapping sounds). Currently, little is known about the neutral substrates underlying these experiences. In this study, 14 participants who experience ASMR, along with 14 control participants, were presented with four video stimuli and four auditory stimuli. Half of these stimuli were designed to elicit ASMR and half were non-ASMR control stimuli. Brain activity was measured using a 32-channel EEG system. The results indicated that ASMR stimuli-particularly auditory stimuli-elicited increased alpha wave activity in participants with self-reported ASMR, but not in matched control participants. Similar increases were also observed in frequency bands associated with movement (gamma waves and sensorimotor rhythm). These results are consistent with the reported phenomenology of ASMR, which involves both attentional and sensorimotor characteristics.

Prenatal low-dose penicillin results in long-term sex-specific changes to murine behaviour, immune regulation, and gut microbiota.

Growing evidence suggests that environmental disruptors of maternal microbes may have significant detrimental consequences for the developing fetus. Antibiotic exposure during early life can have long-term effects on neurodevelopment in mice and humans. Here we explore whether exposure to low-dose penicillin during only the last week of gestation in mice has long-term effects on offspring behaviour, brain, immune function, and gut microbiota. We found that this treatment had sex-specific effects in the adult mouse offspring. Female, but not male, mice demonstrated decreased anxiety-like behaviours, while male, but not female, mice had abnormal social behaviours which correlated with altered brain expression of AVPR1A, AVPR1B, and OXTR, and decreases in the balance of splenic FOXP3+ regulatory T cells. Prenatal penicillin exposure also led to distinct microbiota compositions that clustered differently by sex. These data suggest that exposure of pregnant mice to even a low dose of penicillin through only the last week before birth is nonetheless sufficient to induce long-term sex-specific developmental changes in both male and female offspring.

Antibiotics and the nervous system: More than just the microbes?

The use of antibiotics has recently risen to prominence in neuroscience due to their potential value in studying the microbiota-gut-brain axis. In this context they have been largely employed to illustrate the many influences of the gut microbiota on brain function and behaviour. Much of this research is bolstered by the abnormal behaviour seen in germ-free animals and other well-controlled experiments. However, this literature has largely failed to consider the neuroactive potential of antibiotics themselves, independent from, or in addition to, their microbicidal effects. This is problematic, as clinical as well as experimental literature, largely neglected through the past decade, has clearly demonstrated that broad classes of antibiotics are neuroactive or neurotoxic. This is true even for some antibiotics that are widely regarded as not absorbed in the intestinal tract, and is especially concerning when considering the highly-concentrated and widely-ranging doses that have been used. In this review we will critically survey the clinical and experimental evidence that antibiotics may influence a variety of nervous system functions, from the enteric nervous system through to the brain and resultant behaviour. We will discuss substantial evidence which clearly suggests neuro-activity or -toxicity by most classes of antibiotics. We will conclude that, while evidence for the microbiota-gut-brain axis remains strong, clinical and experimental studies which employ antibiotics to probe it must consider this potential confound.

Immunogenicity of bacteriophages.

Hundreds of trillions of diverse bacteriophages (phages) peacefully thrive within and on the human body. However, whether and how phages influence their mammalian hosts is poorly understood. In this review, we explore current knowledge and present growing evidence that direct interactions between phages and mammalian cells often induce host inflammatory and antiviral immune responses. We show evidence that, like viruses of the eukaryotic host, phages are actively internalized by host cells and activate conserved viral detection receptors. This interaction often generates proinflammatory cytokine secretion and recruitment of adaptive immune programs. However, significant variability exists in phage-immune interactions, suggesting an important role for structural phage characteristics. The factors leading to the differential immunogenicity of phages remain largely unknown but are highly influenced by their human and bacterial hosts.

The Effect of Microbiota on Behaviour.

There is currently enormous interest in the impact of the intestinal microbiota on the development and function of the brain via activity of the microbiota-gut-brain axis. It has long been recognised that symbiotic microorganisms influence host behaviour, but in recent years evidence has accumulated that this can, in fact, be beneficial to the host. Indeed, substantial research has now demonstrated an influence of the intestinal microbiota on a wide range of mammalian behaviours. Here, we review what is currently known about the influence of intestinal microbiota on learning and memory, olfaction, social behaviours, and circadian processes. While work in animal models is compelling, further work is required to elucidate mechanisms whereby bacterial influence is occurring, as well as to determine the extent to which gut microbiota can influence similar phenotypes in humans.

Membrane vesicles of Lacticaseibacillus rhamnosus JB-1 contain immunomodulatory lipoteichoic acid and are endocytosed by intestinal epithelial cells.

Intestinal bacteria have diverse and complex influence on their host. Evidence is accumulating that this may be mediated in part by bacterial extracellular membrane vesicles (MV), nanometer-sized particles important for intercellular communication. Little is known about the composition of MV from gram-positive beneficial bacteria nor how they interact with intestinal epithelial cells (IEC). Here we demonstrate that MV from Lacticaseibacillus rhamnosus JB-1 are endocytosed in a likely clathrin-dependent manner by both mouse and human IEC in vitro and by mouse IEC in vivo. We further show that JB-1 MV contain lipoteichoic acid (LTA) that activates Toll-like receptor 2 (TLR2) and induces immunoregulatory interleukin-10 expression by dendritic cells in an internalization-dependent manner. By contrast, neither LTA nor TLR2 appear to be required for JB-1 MV endocytosis by IEC. These results demonstrate a novel mechanism by which bacterial MV can influence host physiology and suggest one potential route for beneficial influence of certain bacteria and probiotics.

Bacterial membrane vesicles and phages in blood after consumption of lacticaseibacillus rhamnosus JB-1.

Gut microbiota have myriad roles in host physiology, development, and immunity. Though confined to the intestinal lumen by the epithelia, microbes influence distal systems via poorly characterized mechanisms. Recent work has considered the role of extracellular vesicles in interspecies communication, but whether they are involved in systemic microbe-host interaction is unclear. Here, we show that distinctive nanoparticles can be isolated from mouse blood within 2.5 h of consuming Lacticaseibacillus rhamnosus JB-1. In contrast to blood nanoparticles from saline-fed mice, they reproduced lipoteichoic acid-mediated immune functions of the original bacteria, including activation of TLR2 and increased IL-10 expression by dendritic cells. Like the fed bacteria, they also reduced IL-8 induced by TNF in an intestinal epithelial cell line. Though enriched for host neuronal proteins, these isolated nanoparticles also contained proteins and viral (phage) DNA of fed bacterial origin. Our data strongly suggest that oral consumption of live bacteria rapidly leads to circulation of their membrane vesicles and phages and demonstrate a nanoparticulate pathway whereby beneficial bacteria and probiotics may systemically affect their hosts.

The vagus nerve is necessary for the rapid and widespread neuronal activation in the brain following oral administration of psychoactive bacteria.

There is accumulating evidence that certain gut microbes modulate brain chemistry and have antidepressant-like behavioural effects. However, it is unclear which brain regions respond to bacteria-derived signals or how signals are transmitted to distinct regions. We investigated the role of the vagus in mediating neuronal activation following oral treatment with Lactobacillus rhamnosus (JB-1). Male Balb/c mice were orally administered a single dose of saline or a live or heat-killed preparation of a physiologically active bacterial strain, Lactobacillus rhamnosus (JB-1). 165 min later, c-Fos immunoreactivity in the brain was mapped, and mesenteric vagal afferent fibre firing was recorded. Mice also underwent sub-diaphragmatic vagotomy to investigate whether severing the vagus prevented JB-1-induced c-Fos expression. Finally, we examined the ΔFosB response following acute versus chronic bacterial treatment. While a single exposure to live and heat-killed bacteria altered vagal activity, only live treatment induced rapid neural activation in widespread but distinct brain regions, as assessed by c-Fos expression. Sub-diaphragmatic vagotomy abolished c-Fos immunoreactivity in most, but not all, previously responsive regions. Chronic, but not acute treatment induced a distinct pattern of ΔFosB expression, including in previously unresponsive brain regions. These data identify that specific brain regions respond rapidly to gut microbes via vagal-dependent and independent pathways, and demonstrate that acute versus long-term exposure is associated with differential responses in distinct brain regions.

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.

LRRTMs Organize Synapses through Differential Engagement of Neurexin and PTPσ.

Presynaptic neurexins (Nrxs) and type IIa receptor-type protein tyrosine phosphatases (RPTPs) organize synapses through a network of postsynaptic ligands. We show that leucine-rich-repeat transmembrane neuronal proteins (LRRTMs) differentially engage the protein domains of Nrx but require its heparan sulfate (HS) modification to induce presynaptic differentiation. Binding to the HS of Nrx is sufficient for LRRTM3 and LRRTM4 to induce synaptogenesis. We identify mammalian Nrx1γ as a potent synapse organizer and reveal LRRTM4 as its postsynaptic ligand. Mice expressing a mutant form of LRRTM4 that cannot bind to HS show structural and functional deficits at dentate gyrus excitatory synapses. Through the HS of Nrx, LRRTMs also recruit PTPσ to induce presynaptic differentiation but function to varying degrees in its absence. PTPσ forms a robust complex with Nrx, revealing an unexpected interaction between the two presynaptic hubs. These findings underscore the complex interplay of synapse organizers in specifying the molecular logic of a neural circuit.

Oral selective serotonin reuptake inhibitors activate vagus nerve dependent gut-brain signalling.

The vagus nerve can transmit signals to the brain resulting in a reduction in depressive behavior as evidenced by the long-term beneficial effects of electrical stimulation of the vagus in patients with intractable depression. The vagus is the major neural connection between gut and brain, and we have previously shown that ingestion of beneficial bacteria modulates behaviour and brain neurochemistry via this pathway. Given the high levels of serotonin in the gut, we considered if gut-brain signaling, and specifically the vagal pathway, might contribute to the therapeutic effect of oral selective serotonin reuptake inhibitors (SSRI). Mesenteric nerve recordings were conducted in mice after treatment with SSRI to ascertain if this class of drugs resulted in increased vagal excitability. Patch clamp recordings of enteric neurons were carried out to measure activity of primary afferent neurons in the gut in response to SSRI and to assess the importance of gut epithelium in transducing signal. The tail suspension test (TST) was used following 14d feeding of SSRI in vagotomised and surgical sham mice to measure depressive-like behaviour. Brain mRNA expression was examined via PCR and the intestinal microbiome was assessed. Mesenteric nerve recordings in BALB/c mice demonstrated that oral treatment with SSRI leads to a significant increase in vagal activity. This effect was not observed in mice treated with a representative noradrenaline-dopamine reuptake inhibitor. It is known that signals from the gut can be transmitted to the vagus via the enteric nervous system. Exposure of the gut to SSRI increased the excitability of intrinsic primary afferent neurons in the myenteric plexus, through an intestinal epithelium dependent mechanism, and alpha-diversity of gut microbiota was altered. Critically, blocking vagal signaling from gut to brain, via subdiaphragmatic vagotomy, abolished the antidepressive effects of oral SSRI treatment as determined by the tail suspension test. This work suggests that vagus nerve dependent gut-brain signaling contributes to the effects of oral SSRI and further, highlights the potential for pharmacological approaches to treatment of mood disorders that focus on vagal stimulation and may not even require therapeutic agents to enter the circulation.

Low-Density Primary Hippocampal Neuron Culture.

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This 'sandwich culture' allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.