The Gut-Brain-Axis & Anxiety

Vagus Nerve Pathway

Human genomics and genome sequencing have piloted treatment opportunities and patient care with precision and individualized medicine. One of the peak questions of the field of microbiome research is could our knowledge of the human microbiota be integrated in medicine in a similar fashion? ¹⁹ A mountain of recent evidence has revealed gut microbes influence health and disease, where variations in microbiota composition can regulate normal physiology and contribute to disorders in the CNS. The microbiota-gut-brain axis is at the intersection and balance of neurodevelopment and mental illness. Multiple endocrine, immune, and neural pathways relay signals and information throughout the gut-brain axis. ¹² The vagus nerve is a bidirectional, continuous pathway from the gut to the brain.

The data presented below by Bravo and colleagues looks at the importance of the vagus pathway between the gut-microbiome and the brain, presenting how this nerve impacts brain function and behavior ¹. Bravo and colleagues found a GABA mediated bacteria (L.rhamnosus) can decrease anxiety and depressive-like behaviors in mouse models. This is a pivotal study in not only understanding how bacteria can regulate CNS function in mice, but also downstream communication of stress as induced within the microbiota, and the use of a vagotomy in microbiome research. Here, the impact of microbiota on neurotransmission, anxiety-like and depressive-like behavior is expanded on through understanding a key signaling pathway of this axis.

The Vagus Nerve

Figure 1. Bidirectional communication between the gut microbiota and the central nervous system through the vagus nerve (VN). Gut endocrine cells stimulate vagus afferent fibers, which can signal the central autonomic network (CAN). VN afferent fibers stimulate efferent fibers to reduce inflammation in the digestive tract, intestinal permeability, and further interactions in the gut microbiota. Image from Bonaz et al. (2018) ⁷

The vagus nerve (Cranial Nerve X) is the longest nerve in the human body, and compromises a principal role in the autonomic peripheral nervous system. It's a twofold nerve comprised of sensory (afferent) and motor (efferent) fibers. Somatic and visceral sensations felt on our skin and internal organs are distributed from this nerve. It travels from the medulla in the brainstem to the cervical liver, chest, abdomen, and to the colon. Activating vagal neurons relays signals from the gastrointestinal tract to the brainstem, mediating anxiety and depressive-related behaviors.

Perhaps the fastest highway for communication between the gut and the brain, is how microbiota "hijack" vagus nerve signaling. ¹² The data presented by Bravo et al., situates the vagus nerve at the interface of gut-brain communication and maintaining homeostasis within the autonomic NS and the microbiome.

Measuring Anxiety and Depressive-like Behavior in Mouse Models

GABA

GABA is the main inhibitory neurotransmitter in the CNS, mediated by ionotropic GABA_A receptors and metabotropic GABA_B receptors. GABA_A , with subunits α, β, and γ, and GPCR GABA_B ,with subunits GABA_B1 and GABA_B2 are both used in drugs and pharmaceutical interventions. Alterations in GABA_A and GABA_B receptor agonists are important in stress-related psychological disorders of anxiety and depression.

L. rhamnosus (JB-1)

L. rhamnosus (JB-1) is a probiotic bacteria found in the commensal microbiota, and has been tested to regulate stress, anxiety, and irritate bowl syndrome (IBS). Bravo and colleagues sought to understand if this bacteria could alter brain function in mice and subsequent GABA neurotransmission. Bravo et al., administered L. rhamnosus (JB-1) in mice to assess alterations in GABA expression, anxiety, and depressive-like behavior.

Vagotomy (Vx)

Bravo et al., performed a subdiaphragmatic vagotomy (Vx), to investigate the communication of sensory information from the gut microbiota to the brain and it's impact on GABA expression. A vagotmy is a surgical technique to remove all vagal fibers: afferent and efferent fibers ¹⁰. The Sham surgery is a placebo surgery used to neutralize bias in animal models. Broth-fed and Sham surgery controls were implemented to compare the effects of L. rhamnosus (JB-1) and a vagotomy on behavior.

They assessed the effects of Vx on broth fed mice and L.rhamnosus (JB-1) treated mice, by measuring time in the center of a open field arena, # entries to the center, and time immoble in a forced swim test. They compared the Vx/broth and Vx/ L.rhamnosus (JB-1) with Sham/broth controls and Sham/L.rhamnosus (JB-1) treated mice (n=10).

The Open Field Test

Bravo et al., administed an open field test to measure anxiety-related behavior in mice. This test is used to measure behavior, locomotor activity, and exploration patterns in mice models. Mice were placed in an open field for 10 minutes and their total distance traveled, speed, and time spent in the center of the field were recorded. More time spent in the outer edges of the arena, less time in the center, indicates increased anxiety. ⁶

Bravo et al., implemented a forced swim test (FST) test to measure depressive like behavior in mice. In the FST mice were placed in a glass cylinder filled with water and for a 6 minute test/4 minute observation period their activity and mobility was recorded. A mouse was considered ‘immobile’ when their behaviors was just sufficient enough to keep their head above water, thus measuring depressive -related behavior and chronic stress related to locomotion. ²³

Figure 2. Open Field Test. Images taken from Stanford Behavioral and Functional Neuroscience Laboratory. ⁶

Effects of a Vagotomy on Brain & Behavior

Figure 3A. Vagotomy (Vx) prevents the effects of L.rhamnosus (JB-1) in mediating anxiety and depressive-like behaviors. Significance denoted with *. More time and entries in the central area of the open field is indicative of decreased anxiety-like behavior. (A) Sham/L.rhamnosus (JB-1) treated mice (n=10) (white bars) spent more time in the central area of an open field arena in comparison to Sham/broth fed animals (n=10) (black bars). Sham/L.rhamnosus (JB-1) treated mice (n=10) had significantly more entries in the center of the area than Sham/broth treated mice (n=10) These behaviors were prevented by Vx, as the Vx/broth and Vx/L.rhamnosus (JB-1) treated mice both had significantly less time and entries in the center arena. There was no significant difference of distance traveled (motor activity) between the control and experimental groups, implying the differences in time and entries in the center arena are not due to an effect of locomotion.

Figure 3B and 3C. Vx prevents the effect s of L. rhamnosus (JB-1) on GABA_Aα2 & GABA_Aα₁mRNA expression in the dentate gyrus (DG), cornus ammonia region 3 (CA3), and cornus ammonia region 1 (CA1). (B) Sham/L.rhamnosus (JB-1) mice have significantly higher levels of GABA_Aα2 mRNA expression in the DG and CA3 areas in comparison with sham/broth animals (n = 6). Vx prevents the effect of L.rhamnosus (JB-1) on hippocampal GABA_Aα2 mRNA expression in DG CA3 and CA1. (C) Sham/L.rhamnosus (JB-1) (n=6) mice showed significantly lower levels of GABA_Aα1 mRNA in DG, CA3, and CA1 regions compared to broth fed animals. Vx prevented any effect of L.rhamnosus (JB-1) on hippocampal GABA_Aα1 mRNA in the GD, CA3, and CA1 regions in both experimental groups.

Implications

Anxiety & Depression

Mice spent significantly more time and exhbited more entries in the center of the open field from the injection of L.rhamnosus (JB-1). As the motor activity in each experimental group was constant, the authors can attribute this exploratory behavior from L.rhamnosus (JB-1) to it's anxiolytic (anti-anxiety) effects. After a vagotomy, these results were inhbited for both groups treated with either broth or L.rhamnosus (JB-1). The reduction in anxiety and depressive-like behavioral effects of L. rhamnosus (JB-1), as shown by the open field and forced swim test, are prevented by the vagotomy. (Fig. 3A) Anxiety and depressive-related behaviors are mediated by the vagus nerve.

GABAergic System

Increased levels of GABA_Aα2in the DG, CA3, and CA1 regions from L.rhamnosus (JB-1) were prevented by Vx. (Fig 3B) Similarly, decreased levels of GABA_Aα₁in the DG, CA3, and CA1 regions were prevented by Vx. (3C) GABA_Aα2 & GABA_Aα₁ mRNA were consistently modulated by L.rhamnosus (JB-1). GABA_Aα2has been shown to regulate the anti-anxiety effects of benzodiazepines, where GABA_Aα₁regulates it's depressant effects. (Bravo, 2011) The behavioral findings, of how L.rhamnosus (JB-1) showed anti anxiety and antidepressant related behaviors, it supported by the expression of GABA_Aα2andGABA_Aα₁in the hippocampal regions DG, CA1, and CA3.

Vagus Nerve

Following Bravo et al., multiple studies have tested the effects of a vagotomy on mice, such as the infleunce of the vagus nerve in signaling a bacteria L. reuteri to mediate social deficits in mouse models ²². Bravo et al., demonstrated a direct pathway through the vagus nerve in communicating between bacteria in the gut and the brain. This was a landmark study in understanding the importance of the vagus nerve and it's role in relaying information throughout the periphery and influencing CNS function. In finding how bacteria modulate the GABAergic system, they have presented possible theraputic targets for psychiatric disorders.

Outstanding Questions

As this study implemented a vagotomy (surgically removed all afferent and efferent vagus fibers) how would the effect on brain and behavior change with a dysbiosis in the vagus pathway, for example in someone with partially nonfunctioning vagus fibers or a nonmyelinated nerve?

As this study observes neurological changes in GABA subunit expression at the mRNA level, could future research target changes in protein transcripts for a more complex look at signaling cascades?

How can further research expand on the procedure put forth by Bravo and colleagues to stimulate more complex microbiota and neurodevelopmental interactions often found in individualized medicine?

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