of 8
The gut microbiota–brain axis in behaviour
and brain disorders
In the format provided by the
authors and unedited
Supplementary information
https://doi.org/10.1038/s41579-020-00460-0
Supplementary Table 1. Approaches and Tools for Investigating the Gut microbiota
-
brain
axis
Technique/tool
Principle
Refs. to
Microbiota
R
esearch
Other
Refs.
16S microbiome
profiling
Sequencing of the 16S rRNA gene isolated from gut
samples (either
tissue or fecal samples) is used to provide
the general composition of the bacteria in the gut from a
“bird’s eye” perspective (i.e., roughly at the genus level
and higher taxonomy). This information is a useful starting
point for gut microbiota investigat
ions.
1
5
Shotgun
metagenomic
sequencing
Provides a comprehensive description of gut microbial
communities at the species level as well as the
gene
catalog
carried by those members through sequencing of
DNA isolated from the gut microbiota. Can be used to
define the “functional potential” of a
polymicrobial
community (e.g., their metabolic complexity, presence of
classical virulence genes,
etc.
).
6
8
Shotgun
metatranscriptomic
sequencing
Provides survey of gut microbiome transcriptional activity,
which acts as a proxy for bacterial physiology.
This RNA
sequencing approach s
hould be used in conjunction with
metagenomic
(DNA)
sequencing to provide context to
expression patterns, and is has b
een applied in chronic
diseases, such as inflammatory bowel disease.
9
Metabolomics
Provides survey of molecules, nutrient sources, and other
metabolic pr
oducts that are available in different body
compartments, including the gut, brain, and circulatory
systems.
10,11
In vitro
bacterial
culturing
Culturing of bacteria
in vitro
allows for fine control of the
bacteria’s environment, which can be used to determine
12,13
bacterial metabolism (including the degradation of
psychiatric drugs) and cooperative growth
using signaling
molecules.
Germ
-
free and
antibiotic treated
mouse models
Provides ability to test necessity of gut microbiota in
mediating
biological
effects, as removal of the gut
microbiota can be tested to either enhance or suppress
phenotypes. Germ
-
free animals must be
bred and
maintained
under completely sterile conditions
, but
antibiotic administration has been used to study
depletion
of
gut
bacteria
from animals born with a complete
microbiota.
14
18
Fecal microbiota
transfers in mice
(not clinical FMT)
The gut microbiota
of mammals (e.g., humans or mice,
etc.) can be transferred to germ
-
free or antibiotic treated
rodents through gavage with slurries from donor fecal
samples. This can be used to test
i
f the gut microbiota is
sufficient to transfer a phenotype from one mouse
to
another or from a human to a
recipient mouse
.
1,2,10,19
Monocolonization
The colonization of a germ
-
free mouse with a single
bacterial species to determine their sufficien
cy in
modulating a phenotype. Can be expanded to include
gene
knockouts of specific pathways in genetically tractable
organisms.
11,20
Probiotic
administration
Bacteria can be administered to rodent models through
gavage or, in some cases, through drinking water to
suppress adverse neuropsychiatric phenotypes. Stable
colonization of probiotics is difficult to attain given the
nature of colonization resistance
by
the gut microbiota, so
probiotics may require chronic administration to have an
appreciable effect.
21,22
Gene knockouts (KO)
and transgenic
Useful for studying genes and genetic pathways implicated
disease or other outcomes
. KO and transgenic animals can
11,17,18,23
animals
be used for modelling
human diseases
associat
ed to
genetic risks.
Cre and/or Lox
-
P
recombinase animals
Site, cell type and time
-
specific recombinati
on system
widely used for conditional gene
-
targeting
, a
bioengineering tool repurposed from bacteriophage
recombination systems.
23,24
CRISPR/Cas9 system
Cas9 has been used to induce cell
-
type (e.g., neurons)
genome
-
editing and generation of stable gene knockouts
, a
bioengineering tool adapted from bacterial “immune
systems” to phage infection.
Useful to model disorders
associated to gene
tic risks, including ASD.
25
Pharmacological
agents
Drugs
for s
tudying neurocircuitry and neurotransmitter
involvement. Can have limited site and time specificity
(e.g., 5
-
HT receptor agonists, neurotransmitter receptor
antagonists, or sodium channel blockers).
19,23,26
Vagotomy
The surgical severance of the vagus nerve
that disrupts
signaling from various peripheral organs to the brain
.
Different types of vagotomy can be
used depending on the
goal (e.g., truncal vagotomy, selective vagotomy).
21,27,28
Optogenetics
Enables
genetically engineered cells to express membrane
bound light
-
sensitive proteins (opsins) that can affect
neuronal activity, inhibition, or modulation of intracellular
signaling in transgenic animals (e.g., Cre
-
recombinase
animals).
Activity turned on or o
ff by light.
N/A
29
Chemogenetics
E
ngineered proteins are used to reach neuronal temporal
and spatial specificity in transgenic animals. Des
igner
receptors exclusively activated by designer drugs
(DREADDs) are the most common tool used to define
neuronal population activity.
30
31
Electrophysiology
Technique that explores electrical activity from action
potentials in neuronal cells
in vitro
and
in vivo
(live
10,22,23
behaving animals).
Golgi staining, Dyes
and Tracing Proteins
Tracing tools that can be deliver to nervous system cells
(e.g., non
-
specific membrane tracing; Fluorogold and
Retrobeads; dextran amines).
32
Immediate Early
Genes (IEG) readouts
Allows a broad
overview of recently activated neurons
during defined window
s
of time and in response to a
specific
stimulus.
33
Functional Magnetic
Resonance (fMRI)
Whole brain imaging in a live subject for functional
connectivity measurements. Poor temporal resolution. Not
cell
-
type specific.
34,35
Electron microscopy
Allows ultrastructure
cell morphology and visualization of
fine synapsis connectivity.
36,37
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