More Fun With the Gut Microbiota and the Brain (In Mice)

Everyone has been tweeting this new paper, and I have to say, it's a doozy.  I don't recommend that many papers, as most are fairly painful to read (though many papers are full of interesting facts, they don't teach scientists how to write), but if you can get your hands on this one, please do so.

The Intestinal Microbiota Affect Central Levels of Brain-Derived Neurotropic Factor and Behavior in Mice

Let's dive in.  Ooh, I like the first part:

The intestinal microbiota is a vast ecosystem that shapes a wide variety of host functions, both within and outside the gastrointestinal tract.

Y'all may recall my previous article in which we have evidence that your commensal beasties, which make up 90% of your cells, also control a mouse's brain (and possibly yours.)  And something like this paper and this experimental design excite me more than the endocannabinoid/rat paper I wrote about yesterday.  I don't doubt that we probably have endocannabinoid receptors for corn oil (which may be activated by all kinds of fats) in our guts, or ones for sweets in our mouths, I'm just not sure what it means when isolated in that way via the sham-feeding and with creepy rat chow liquid diets that immediately drain out and dribble onto the rat cage in a gross gut slurry.  However, we do have to keep in mind that these are rodents who diverged from the family tree many many many millions of years ago…

Back to beasties.  They have found that colonization of germ-free mice with Bacteroides species affect the expression of messenger RNA that encode for immune and smooth muscle function, gut permeability, and the gut nervous system.  Regulation of body weight and pain perception in the skin have also been shown to be affected by changes in microbiota.

In healthy folks, the intestinal microbiota are diverse, and the species relatively stable.  In conditions such as inflammatory bowel disease (Crohn's and ulcerative colitis => IBD) and in irritable bowel syndrome (IBS), the beastie species are less diverse and tend to be unstable over time.  In both IBD and IBS, depression and anxiety are common, and associated with worse gastrointestinal disease.  In IBS, studies have shown 50-90% of the people affected also have psychiatric symptoms.   But are the psychiatric symptoms caused by the discomfort and stress of living with irritable or inflammatory bowel conditions, or are the symptoms caused by the same pathology that causes the gut issues - which includes alteration in the intestinal microbiota.

Previous experimental evidence that gut bacteria affect the brain includes:

1. In mice, microbiota have been shown to affect the hypothalamic-pituitary response to stress.
2. Oral antibiotics seem to help people suffering from neurological complications of end-stage liver disease.
3. Pathogenic bacteria introduced into the gut causes anxiety-like behavior in mice.

For this experiment, the researchers obtained some regular and germ-free mice who lived thereafter in level II biocontainment.  Some of the germ-free mice remained germ-free.  Others got fecal transplants from other mice that had known bacterial species on board.  Some of the mice got a whopping dose of antibiotics (that were "nonabsorbable" so presumably only affected the gut microbiota - a separate experiment by which a small amount of antibiotics were injected systemically in some mice acted as a back-up control and did not affect the gut microbiota).  Control mice lapped up sterile water instead.

The mice's gut contents were extracted, cultured, and the bacterial DNA and RNA were characterized via polymerase chain reaction and gel electrophoresis, so you could see what kind of beasties survived the various treatments in each experimental and control group of mice.  The mice also underwent behavioral testing - how open they were to exploration, how quick they were to step off a raised platform, etc.

In addition (these researchers were pretty darn thorough) - the gut linings were examined for signs of inflammation, and cytokines and neurotransmitter levels in the small intestine and colon were measured.  The brains (specifically sections of the hippocampus and amygdala) were extracted and measured for levels of brain-derived neurotrophic factor (BDNF).  BDNF is a brain fertilizer whose levels in the hippocampus and amygdala correlate with depression and anxiety symptoms.  Low BDNF in the hippocampus is generally bad news and are associated with depression and anxiety symptoms.  Antidepressants are thought to work by increasing levels of BDNF there.  In the amygdala, the fear center of the brain, lower levels of BDNF seem to correlate with less fear.  

Still with me?  How about a song.  The Strokes - Taken For A Fool (right click to open in new tab or window if you don't want to be shipped away from this page.)

Results.  Well, whopping antibiotic administration did affect the beastie species in the guts of the mice.  Lactobacilli and Actinobacteria populations increased with the treatment,  while the Bacteroides and gamma-proteobacteria decreased (including Shigella and Klebsiella).  The mice who had been exposed to antibiotics were more exploratory and less apprehensive than the control mice.  After two weeks, however, the behavioral testing of the control and antibiotic-treated mice when back to being the same, and measures of their beastie populations showed they went back to normal.  In the germ-free mice, antibiotic administration did nothing to affect their behavior, showing once again it was likely the alterations in the beastie population that caused the behavioral change in the experimental group.  And, finally, germ-free mice who got the fecal transplants showed changes in behavior after being colonized compared to the germ-free controls.

So you see, these researchers went backwards, forwards, and sideways to prove their findings.  Pretty cool, really.

More results.  Mice who got the whopping dose of antibiotics had higher levels of BDNF in the hippocampus and lower amounts of BDNF in the amygdala compared to control mice.  These BDNF levels are what you would expect if you have less anxious and fearful mice who are more open to exploring than controls.  The antibiotic-treated mice did not have any changes of cytokines in their guts to suggest increased inflammation, or any increased pathologic inflammatory markers under microscope, and there were no changes in gut neurotransmitters.  SO - the behavior and BDNF changes had nothing to do with gut inflammation or the enteric nervous system (at least involving the neurotransmitters they tested, which included all the biggies).

The results of this study provide strong evidence for a microbiota-gut-brain-axis that influences brain biochemistry and modulates behavior in adult mice.

In another paper, researchers showed that mice lacking a gut sympathetic nervous system (via severing the vagus nerve or using chemical neurotoxins that kill the sympathetic nervous system) were not affected by the addition of Campylobacter (a gut pathogen) to the gut, which induced anxiety in normal mice.   Thus the anxiety behavior seemed to be mediated via communication between the gut and brain via the vagus nerve.  In the current paper, a group of mice who had a severed vagus nerve and another group who had been exposed to the neurotoxin had the same results as the mice with untouched nervous systems, thus the change in behavior and changes in BDNF in the brain brought on by antibiotic treatment in the gut occurred through another mechanism - the researchers thought substances produced by the gut bacteria likely acted directly or indirectly on the central nervous system somehow.

Another recent paper showed that mice fed a beef-enriched diet for three months in early childhood (mouse puppyhood?) had improved memory compared to rats fed standard rat chow.  (I'm not sure exactly if that can be linked entirely to the gut bacteria, but it is mentioned in this paper and I thought it was a cool fact.)

These researchers have already shown that administration of probiotics could normalize behavior in mice who showed alterations in behavior and mild to moderate gut inflammation.

All told, I think these scientists are beginning to elucidate a pretty fascinating area of interest to psychiatrists, gastroenterologists, and mouse enthusiasts everywhere.