Microbiota-modulated CART+ enteric neurons autonomously regulate blood glucose.

The gut microbiota affects tissue physiology, metabolism, and function of both the immune and nervous systems. We found that intrinsic enteric-associated neurons (iEANs) in mice are functionally adapted to the intestinal segment they occupy; ileal and colonic neurons are more responsive to microbial colonization than duodenal neurons. Specifically, a microbially responsive subset of viscerofugal CART+ neurons, enriched in the ileum and colon, modulated feeding and glucose metabolism. These CART+ neurons send axons to the prevertebral ganglia and are polysynaptically connected to the liver and pancreas. Microbiota depletion led to NLRP6- and caspase 11-dependent loss of CART+ neurons and impaired glucose regulation. Hence, iEAN subsets appear to be capable of regulating blood glucose levels independently from the central nervous system.

Author Correction: Microbiota modulate sympathetic neurons via a gut-brain circuit.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Microbiota modulate sympathetic neurons via a gut-brain circuit.

Connections between the gut and brain monitor the intestinal tissue and its microbial and dietary content1, regulating both physiological intestinal functions such as nutrient absorption and motility2,3, and brain-wired feeding behaviour2. It is therefore plausible that circuits exist to detect gut microorganisms and relay this information to areas of the central nervous system that, in turn, regulate gut physiology4. Here we characterize the influence of the microbiota on enteric-associated neurons by combining gnotobiotic mouse models with transcriptomics, circuit-tracing methods and functional manipulations. We find that the gut microbiome modulates gut-extrinsic sympathetic neurons: microbiota depletion leads to increased expression of the neuronal transcription factor cFos, and colonization of germ-free mice with bacteria that produce short-chain fatty acids suppresses cFos expression in the gut sympathetic ganglia. Chemogenetic manipulations, translational profiling and anterograde tracing identify a subset of distal intestine-projecting vagal neurons that are positioned to have an afferent role in microbiota-mediated modulation of gut sympathetic neurons. Retrograde polysynaptic neuronal tracing from the intestinal wall identifies brainstem sensory nuclei that are activated during microbial depletion, as well as efferent sympathetic premotor glutamatergic neurons that regulate gastrointestinal transit. These results reveal microbiota-dependent control of gut-extrinsic sympathetic activation through a gut-brain circuit.

Adrenergic Signaling in Muscularis Macrophages Limits Infection-Induced Neuronal Loss.

Enteric-associated neurons (EANs) are closely associated with immune cells and continuously monitor and modulate homeostatic intestinal functions, including motility and nutrient sensing. Bidirectional interactions between neuronal and immune cells are altered during disease processes such as neurodegeneration or irritable bowel syndrome. We investigated the effects of infection-induced inflammation on intrinsic EANs (iEANs) and the role of intestinal muscularis macrophages (MMs) in this context. Using murine models of enteric infections, we observed long-term gastrointestinal symptoms, including reduced motility and loss of excitatory iEANs, which was mediated by a Nlrp6- and Casp11-dependent mechanism, depended on infection history, and could be reversed by manipulation of the microbiota. MMs responded to luminal infection by upregulating a neuroprotective program via ß2-adrenergic receptor (ß2-AR) signaling and mediated neuronal protection through an arginase 1-polyamine axis. Our results identify a mechanism of neuronal death post-infection and point to a role for tissue-resident MMs in limiting neuronal damage.

Gut macrophages: key players in intestinal immunity and tissue physiology.

The mammalian gastrointestinal tract harbors a large reservoir of tissue macrophages, which, in concert with other immune cells, help to maintain a delicate balance between tolerance to commensal microbes and food antigens, and resistance to potentially harmful microbes or toxins. Beyond their roles in resistance and tolerance, recent studies have uncovered novel roles played by tissue-resident, including intestinal-resident macrophages in organ physiology. Here, we will discuss recent advances in the understanding of the origin, phenotype and function of macrophages residing in the different layers of the intestine during homeostasis and under pathological conditions.

Compartmentalized gut lymph node drainage dictates adaptive immune responses.

The intestinal immune system has the challenging task of tolerating foreign nutrients and the commensal microbiome, while excluding or eliminating ingested pathogens. Failure of this balance leads to conditions such as inflammatory bowel diseases, food allergies and invasive gastrointestinal infections1. Multiple immune mechanisms are therefore in place to maintain tissue integrity, including balanced generation of effector T (TH) cells and FOXP3+ regulatory T (pTreg) cells, which mediate resistance to pathogens and regulate excessive immune activation, respectively1-4. The gut-draining lymph nodes (gLNs) are key sites for orchestrating adaptive immunity to luminal perturbations5-7. However, it is unclear how they simultaneously support tolerogenic and inflammatory reactions. Here we show that gLNs are immunologically specific to the functional gut segment that they drain. Stromal and dendritic cell gene signatures and polarization of T cells against the same luminal antigen differ between gLNs, with the proximal small intestine-draining gLNs preferentially giving rise to tolerogenic responses and the distal gLNs to pro-inflammatory T cell responses. This segregation permitted the targeting of distal gLNs for vaccination and the maintenance of duodenal pTreg cell induction during colonic infection. Conversely, the compartmentalized dichotomy was perturbed by surgical removal of select distal gLNs and duodenal infection, with effects on both lymphoid organ and tissue immune responses. Our findings reveal that the conflict between tolerogenic and inflammatory intestinal responses is in part resolved by discrete gLN drainage, and encourage antigen targeting to specific gut segments for therapeutic immune modulation.

Can U Turn ILC2s Up?

Innate lymphoid cells (ILCs) are tuned to quickly respond to and amplify tissue-specific signals. Work of three independent groups in Nature uncovers a novel mode of inflammatory communication between ILC2s and neurons at mucosal surfaces.

Spineless Behavior of CX3CR1+ Monocytes in Response to Infection.

Sickness in mammals can lead to cognition deficits, although the underlying mechanisms remain elusive. In a recent Nature Medicine article, Garré et al. (2017) report that sickness-induced cortical dendritic spine loss and impaired memory formation is mediated by CX3CR1+ monocyte-derived TNF-α.

Neuro-immune Interactions Drive Tissue Programming in Intestinal Macrophages.

Proper adaptation to environmental perturbations is essential for tissue homeostasis. In the intestine, diverse environmental cues can be sensed by immune cells, which must balance resistance to microorganisms with tolerance, avoiding excess tissue damage. By applying imaging and transcriptional profiling tools, we interrogated how distinct microenvironments in the gut regulate resident macrophages. We discovered that macrophages exhibit a high degree of gene-expression specialization dependent on their proximity to the gut lumen. Lamina propria macrophages (LpMs) preferentially expressed a pro-inflammatory phenotype when compared to muscularis macrophages (MMs), which displayed a tissue-protective phenotype. Upon luminal bacterial infection, MMs further enhanced tissue-protective programs, and this was attributed to swift activation of extrinsic sympathetic neurons innervating the gut muscularis and norepinephrine signaling to ß2 adrenergic receptors on MMs. Our results reveal unique intra-tissue macrophage specialization and identify neuro-immune communication between enteric neurons and macrophages that induces rapid tissue-protective responses to distal perturbations.

High-quality draft genome sequences of five anaerobic oral bacteria and description of Peptoanaerobacter stomatis gen. nov., sp. nov., a new member of the family Peptostreptococcaceae.

Here we report a summary classification and the features of five anaerobic oral bacteria from the family Peptostreptococcaceae. Bacterial strains were isolated from human subgingival plaque. Strains ACC19a, CM2, CM5, and OBRC8 represent the first known cultivable members of "yet uncultured" human oral taxon 081; strain AS15 belongs to "cultivable" human oral taxon 377. Based on 16S rRNA gene sequence comparisons, strains ACC19a, CM2, CM5, and OBRC8 are distantly related to Eubacterium yurii subs. yurii and Filifactor alocis, with 93.2 - 94.4 % and 85.5 % of sequence identity, respectively. The genomes of strains ACC19a, CM2, CM5, OBRC8 and AS15 are 2,541,543; 2,312,592; 2,594,242; 2,553,276; and 2,654,638 bp long. The genomes are comprised of 2277, 1973, 2325, 2277, and 2308 protein-coding genes and 54, 57, 54, 36, and 28 RNA genes, respectively. Based on the distinct characteristics presented here, we suggest that strains ACC19a, CM2, CM5, and OBRC8 represent a novel genus and species within the family Peptostreptococcaceae, for which we propose the name Peptoanaerobacter stomatis gen. nov., sp. nov. The type strain is strain ACC19a(T) (=HM-483(T); =DSM 28705(T); =ATCC BAA-2665(T)).

Hippocampal immediate early gene transcription in the rat fluid percussion traumatic brain injury model.

Traumatic brain injury (TBI) is one of the leading causes of neurological disability and death in the USA across all age groups, ethnicities, and incomes. In addition to the short-term morbidity and mortality, TBI leads to epilepsy and severe neurocognitive symptoms, both of which are referenced to post-traumatic hippocampal dysfunction, although the mechanisms of such hippocampal dysfunction are incompletely understood. Here, we study the temporal profile of the transcription of three select immediate early gene (IEG) markers of neuronal hyperactivation, plasticity, and injury, c-fos, brain-derived neurotrophic factor (BDNF), and Bax, in the acute period following the epileptogenic lateral fluid percussion injury in a rodent TBI model. We found that lateral fluid percussion injury leads to enhanced expression of the selected IEGs within 24 h of TBI. Specifically, BDNF and c-fos increase maximally 1-6 h after TBI in the ipsilesional hippocampus, whereas Bax increases in the hippocampus bilaterally in this time window. Antagonism of the N-methyl-D-aspartate-type glutamate receptor by MK801 attenuates the increase in BDNF and Bax, which underscores a therapeutic role for N-methyl-D-aspartate-type glutamate receptor antagonism in the acute post-traumatic time period and suggests a value to a hippocampal IEG readout as an outcome after injury or acute therapeutic intervention.

Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov., obligately anaerobic bacteria from the human oral cavity, and emended description of the genus Oribacterium.

Three strictly anaerobic, Gram-positive, non-spore-forming, rod-shaped, motile bacteria, designated strains ACB1(T), ACB7(T) and ACB8, were isolated from human subgingival dental plaque. All strains required yeast extract for growth. Strains ACB1(T) and ACB8 were able to grow on glucose, lactose, maltose, maltodextrin and raffinose; strain ACB7(T) grew weakly on sucrose only. The growth temperature range was 30-42 °C with optimum growth at 37 °C. Major metabolic fermentation end products of strain ACB1(T) were acetate and lactate; the only product of strains ACB7(T) and ACB8 was acetate. Major fatty acids of strain ACB1(T) were C(14 : 0), C(16 : 0), C(16 : 1)ω7c dimethyl aldehyde (DMA) and C(18 : 1)ω7c DMA. Major fatty acids of strain ACB7(T) were C(12 : 0), C(14 : 0), C(16 : 0), C(16 : 1)ω7c and C(16 : 1)ω7c DMA. The hydrolysate of the peptidoglycan contained meso-diaminopimelic acid, indicating peptidoglycan type A1γ. Genomic DNA G+C content varied from 42 to 43.3% between strains. According to 16S rRNA gene sequence phylogeny, strains ACB1(T), ACB8 and ACB7(T) formed two separate branches within the genus Oribacterium, with 98.1-98.6% sequence similarity to the type strain of the type species, Oribacterium sinus. Predicted DNA-DNA hybridization values between strains ACB1(T), ACB8, ACB7(T) and O. sinus F0268 were <70%. Based on distinct genotypic and phenotypic characteristics, strains ACB1(T) and ACB8, and strain ACB7(T) are considered to represent two distinct species of the genus Oribacterium, for which the names Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov. are proposed. The type strains are ACB1(T) ( = DSM 24637(T) = HM-481(T) = ATCC BAA-2638(T)) and ACB7(T) ( = DSM 24638(T) = HM-482(T) = ATCC BAA-2639(T)), respectively.

Suppression of motor cortical excitability in anesthetized rats by low frequency repetitive transcranial magnetic stimulation.

Repetitive transcranial magnetic stimulation (rTMS) is a widely-used method for modulating cortical excitability in humans, by mechanisms thought to involve use-dependent synaptic plasticity. For example, when low frequency rTMS (LF rTMS) is applied over the motor cortex, in humans, it predictably leads to a suppression of the motor evoked potential (MEP), presumably reflecting long-term depression (LTD) -like mechanisms. Yet how closely such rTMS effects actually match LTD is unknown. We therefore sought to (1) reproduce cortico-spinal depression by LF rTMS in rats, (2) establish a reliable animal model for rTMS effects that may enable mechanistic studies, and (3) test whether LTD-like properties are evident in the rat LF rTMS setup. Lateralized MEPs were obtained from anesthetized Long-Evans rats. To test frequency-dependence of LF rTMS, rats underwent rTMS at one of three frequencies, 0.25, 0.5, or 1 Hz. We next tested the dependence of rTMS effects on N-methyl-D-aspartate glutamate receptor (NMDAR), by application of two NMDAR antagonists. We find that 1 Hz rTMS preferentially depresses unilateral MEP in rats, and that this LTD-like effect is blocked by NMDAR antagonists. These are the first electrophysiological data showing depression of cortical excitability following LF rTMS in rats, and the first to demonstrate dependence of this form of cortical plasticity on the NMDAR. We also note that our report is the first to show that the capacity for LTD-type cortical suppression by rTMS is present under barbiturate anesthesia, suggesting that future neuromodulatory rTMS applications under anesthesia may be considered.

Translational neuromodulation: approximating human transcranial magnetic stimulation protocols in rats.

OBJECTIVE: Transcranial magnetic stimulation (TMS) is a well-established clinical protocol with numerous potential therapeutic and diagnostic applications. Yet, much work remains in the elucidation of TMS mechanisms, optimization of protocols, and in development of novel therapeutic applications. As with many technologies, the key to these issues lies in the proper experimentation and translation of TMS methods to animal models, among which rat models have proven popular. A significant increase in the number of rat TMS publications has necessitated analysis of their relevance to human work. We therefore review the essential principles for the approximation of human TMS protocols in rats as well as specific methods that addressed these issues in published studies. MATERIALS AND METHODS: We performed an English language literature search combined with our own experience and data. We address issues that we see as important in the translation of human TMS methods to rat models and provide a summary of key accomplishments in these areas. RESULTS: An extensive literature review illustrated the growth of rodent TMS studies in recent years. Current advances in the translation of single, paired-pulse, and repetitive stimulation paradigms to rodent models are presented. The importance of TMS in the generation of data for preclinical trials is also highlighted. CONCLUSIONS: Rat TMS has several limitations when considering parallels between animal and human stimulation. However, it has proven to be a useful tool in the field of translational brain stimulation and will likely continue to aid in the design and implementation of stimulation protocols for therapeutic and diagnostic applications.

Safety and tolerability of repetitive transcranial magnetic stimulation in patients with pathologic positive sensory phenomena: a review of literature.

BACKGROUND: Repetitive transcranial magnetic stimulation (rTMS) is emerging as a valuable therapeutic and diagnostic tool. rTMS appears particularly promising for disorders characterized by positive sensory phenomena that are attributable to alterations in sensory cortical excitability. Among these are tinnitus, auditory and visual hallucinations, and pain syndromes. OBJECTIVE: Despite studies addressing rTMS efficacy in suppression of positive sensory symptoms, the safety of stimulation of potentially hyperexcitable cortex has not been fully addressed. We performed a systematic literature review and metaanalysis to describe the rTMS safety profile in these disorders. METHODS: Using the PubMed database, we performed an English-language literature search from January 1985 to April 2011 to review all pertinent publications. Per study, we noted and listed pertinent details. From these data we also calculated a crude per-subject risk for each adverse event. RESULTS: One hundred six publications (n = 1815) were identified with patients undergoing rTMS for pathologic positive sensory phenomena. Adverse events associated with rTMS were generally mild and occurred in 16.7% of subjects. Seizure was the most serious adverse event, and occurred in three patients with a 0.16% crude per-subject risk. The second most severe adverse event involved aggravation of sensory phenomena, occurring in 1.54%. CONCLUSIONS: The published data suggest rTMS for the treatment or diagnosis of pathologic positive sensory phenomena appears to be a relatively safe and well-tolerated procedure. However, published data are lacking in systematic reporting of adverse events, and safety risks of rTMS in these patient populations will have to be addressed in future prospective trials.

Contribution of axonal orientation to pathway-dependent modulation of excitatory transmission by direct current stimulation in isolated rat hippocampus.

Transcranial direct current stimulation (tDCS) is a method for modulating cortical excitability by weak constant electrical current that is applied through scalp electrodes. Although often described in terms of anodal or cathodal stimulation, depending on which scalp electrode pole is proximal to the cortical region of interest, it is the orientation of neuronal structures relative to the direct current (DC) vector that determines the effect of tDCS. To investigate the contribution of neural pathway orientation, we studied DCS-mediated neuromodulation in an in vitro rat hippocampal slice preparation. We examined the contribution of dendritic orientation to the direct current stimulation (DCS) neuromodulatory effect by recording field excitatory postsynaptic potentials (fEPSPs) in apical and basal dendrites of CA1 neurons within a constant DC field. In addition, we assessed the contribution of axonal orientation by recording CA1 and CA3 apical fEPSPs generated by stimulation of oppositely oriented Schaffer collateral and mossy fiber axons, respectively, during DCS. Finally, nonsynaptic excitatory signal propagation was measured along antidromically stimulated CA1 axons at different DCS amplitudes and polarity. We find that modulation of both the fEPSP and population spike depends on axonal orientation relative to the electric field vector. Axonal orientation determines whether the DC field is excitatory or inhibitory and dendritic orientation affects the magnitude, but not the overall direction, of the DC effect. These data suggest that tDCS may oppositely affect neurons in a stimulated cortical volume if these neurons are excited by oppositely orientated axons in a constant electrical field.

Measures of cortical inhibition by paired-pulse transcranial magnetic stimulation in anesthetized rats.

Paired-pulse transcranial magnetic stimulation (ppTMS) is a noninvasive method to measure cortical inhibition in vivo. Long interpulse interval (50-500 ms) ppTMS (LI-ppTMS) provokes intracortical inhibitory circuits and can reveal pathologically impaired cortical inhibition in disorders such as epilepsy. Adaptation of ppTMS protocols to rodent disease models is highly desirable to facilitate basic and translational research. We previously adapted single-pulse TMS (spTMS) methods to rats, but ppTMS has yet to be applied. Specifically, whether ppTMS elicits an inhibitory response in rodents is unknown. ppTMS in rats also requires anesthesia, a setting under which the preservation of these measures is undetermined. We therefore tested, in anesthetized rats, whether anesthetic choice affects spTMS-motor-evoked potentials (MEPs), LI-ppTMS in rats, as in humans, elicits intracortical inhibition of the MEP, and rat LI-ppTMS inhibition is acutely impaired in a seizure model. Rats were anesthetized with pentobarbital (PB) or ketamine-atropine-xylazine (KAX) and stimulated unilaterally over the motor cortex while recording bilateral brachioradialis MEPs. LI-ppTMS was applied analogous to human long interval intracortical inhibition (LICI) protocols, and acute changes in inhibition were evaluated following injection of the convulsant pentylenetetrazole (PTZ). We find that spTMS-evoked MEPs were reliably present under either anesthetic, and that LI-ppTMS elicits inhibition of the conditioned MEP in rats, similar to human LICI, by as much as 58 ± 12 and 71 ± 11% under PB and KAX anesthesia, respectively. LI-ppTMS inhibition was reduced to as much as 53% of saline controls following PTZ injection, while spTMS-derived measures of corticospinal excitability were unchanged. Our data show that regional inhibition, similar to human LICI, is present in rats, can be elicited under PB or KAX anesthesia, and is reduced following convulsant administration. These results suggest a potential for LI-ppTMS as a biomarker of impaired cortical inhibition in murine disease models.

Lateralization of forelimb motor evoked potentials by transcranial magnetic stimulation in rats.

OBJECTIVES: To approximate methods for human transcranial magnetic stimulation (TMS) in rats, we tested whether lateralized cortical stimulation resulting in selective activation of one forelimb contralateral to the site of stimulation could be achieved by TMS in the rat. METHODS: Motor evoked potentials (MEP) were recorded from the brachioradialis muscle bilaterally in adult male anesthetized rats (n=13). A figure-of-eight TMS coil was positioned lateral to midline. TMS intensity was increased stepwise from subthreshold intensities to maximal machine output in order to generate input-output curves and to determine the motor threshold (MT) for brachioradialis activation. RESULTS: In 100% of the animals, selective activation of the contralateral brachioradialis, in the absence of ipsilateral brachioradialis activation was achieved, and the ipsilateral brachioradialis was activated only at TMS intensities exceeding contralateral forelimb MT. With increasing TMS intensity, the amplitudes of both the ipsilateral and contralateral signals increased in proportion to TMS strength. However, the input-output curves for the contralateral and ipsilateral brachioradialis were significantly different (p<0.001) such that amplitude of the ipsilateral MEP was reliably lower than the contralateral signal. CONCLUSIONS: We demonstrate that lateralized TMS leading to asymmetric brachioradialis activation is feasible with conventional TMS equipment in anesthetized rats. SIGNIFICANCE: These data show that TMS can be used to assess the unilateral excitability of the forelimb descending motor pathway in the rat, and suggest that rat TMS protocols analogous to human TMS may be applied in future translational research.

In-session seizures during low-frequency repetitive transcranial magnetic stimulation in patients with epilepsy.

Low-frequency repetitive transcranial magnetic stimulation (rTMS) is emerging as a therapeutic tool for patients with intractable epilepsy. Although seizures during treatment have been reported as adverse events in some patients, the nature and severity of seizures that may be provoked by low-frequency rTMS in patients with epilepsy have not been extensively studied. Accordingly, this article documents seizures in patients (n=5) with intractable epilepsy and average seizure frequency greater than one per day who underwent 1-Hz rTMS for seizure suppression. We report three observations in the present case series: (1) in each instance the in-session seizure was typical in semiology to the patient's habitual seizures, (2) the duration of each documented seizure was either the same as or shorter than the patients' baseline seizures, and (3) the overall neurological outcome on follow-up was not affected by the in-session seizures. More data will be required for valid conclusions with respect to safety and tolerability of low-frequency rTMS in patients with epilepsy, but it is noteworthy from our perspective that seizures during rTMS in this series were similar to the patients' habitual seizures, occurred in patients with epilepsy with baseline seizure frequency exceeding one per day, and did not correlate with a poor neurological outcome or with absence of clinical response to rTMS.