A gut-derived metabolite alters brain activity and anxiety
behaviour in mice
Brittany D. Needham
1
,
Masanori Funabashi
2,3
,
Mark D. Adame
1
,
Zhuo Wang
4
,
Joseph
C. Boktor
1
,
Jillian Haney
5
,
Wei-Li Wu
1,6,7
,
Claire Rabut
8
,
Mark S. Ladinsky
1
,
Son-Jong
Hwang
8
,
Yumei Guo
4
,
Qiyun Zhu
9,10
,
Jessica A. Griffiths
1
,
Rob Knight
9,11,12
,
Pamela
J. Bjorkman
1
,
Mikhail G. Shapiro
8
,
Daniel H. Geschwind
5
,
Daniel P. Holschneider
4,13,14
,
Michael A. Fischbach
2
,
Sarkis K. Mazmanian
1
1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA,
91125, USA.
2
Department of Bioengineering and ChEM-H, Stanford University, Stanford, CA 94305, USA.
3
Translational Research Department, Daiichi Sankyo RD Novare Co., Ltd., Tokyo Japan.
4
Dept. of Psychiatry and the Behavioral Sciences, Keck School of Medicine, University of
Southern California, Los Angeles, CA, 90089, USA.
5
Department of Neurology, University of California Los Angeles, Los Angeles, CA, USA.
6
Department of Physiology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
7
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University,
Tainan, Taiwan.
8
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
CA, 91125, USA.
9
Department of Pediatrics, University of California San Diego, La Jolla, CA, USA.
10
School of Life Sciences, Arizona State University, Tempe, AZ 85281, USA.
11
Department of Computer Science and Engineering, University of California San Diego, La Jolla,
CA, USA;
12
Center for Microbiome Innovation, University of California San Diego, La Jolla, CA, USA.
Correspondence and requests for materials should be addressed to Sarkis Mazmanian, sarkis@caltech.edu or Brittany Needham,
bneedham@caltech.edu.
Author contributions:
Conceptualization: BDN, SKM. Methodology: BDN, MF, MDA, ZW, W-LW, JH, MSL, JAG, CR, SJH, DPH.
Formal Analysis: BDN, MDA, ZW, JH. Investigation: BDN, MF, MDA, ZW, W-LW, JCB, CR, JH, SJH, QZ, MSL, YG. Biochemical
pathway investigation and strain engineering: MF, MAF. Gene abundance analysis: QZ, JCB, RK. fUSi imaging: CR, JCB, BDN,
MGS. 2DG analysis: ZW, BDN, YG, DPH. QuantSeq analysis: JH, W-LW, BDN, DHG. Oligodendrocyte characterization: BDN,
MDA, JCB, MSL, JAG. ET: MSL, BDN, MDA. MRI/DTI: SJH, BDN. Animal behavior: BDN, MDA. Resources: PJB, DG, DPH,
MAF, RK, MGS, SKM. Writing original draft: BDN. Writing Review and Editing: BDN, MF, MDA, ZW, W-LW, JH, MSL,
JAG, DPH, MAF, SKM. Visualization: BDN, MF, MDA, ZW, W-LW, JH, JCB, CR, SJH, MSL, MAF. Supervision: SKM. Project
administration: BDN. Funding acquisition: BDN, WLW, PJB, SKM.
Competing interests:
S.K.M. has financial interests in Axial Biotherapeutics. All other authors declare no competing interests related
to this work.
HHS Public Access
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Published in final edited form as:
Nature
. 2022 February ; 602(7898): 647–653. doi:10.1038/s41586-022-04396-8.
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13
Dept. of Neurology, Keck School of Medicine, University of Southern California, Los Angeles,
CA, 90089, USA.
14
Viterbi School of Engineering, Dept. of Biomedical Engineering, University of Southern
California, Los Angeles, CA, 90033, USA.
Abstract
Integration of sensory and molecular inputs from the environment shapes animal behavior. A
major site of exposure to environmental molecules is the gastrointestinal tract, where dietary
components are chemically transformed by the microbiota
1
and gut-derived metabolites are
disseminated to all organs, including the brain
2
. In mice, the gut microbiota impacts behavior
3
,
modulates neurotransmitter production in the gut and brain
4
,
5
, and influences brain development
and myelination patterns
6
,
7
. Mechanisms mediating gut-brain interactions remain poorly defined,
though broadly involve humoral or neuronal connections. We previously reported that levels
of the microbial metabolite 4-ethylphenyl sulfate (4EPS) were elevated in a mouse model of
atypical neurodevelopment
8
. Herein, we identified biosynthetic genes from the gut microbiome
that mediate conversion of dietary tyrosine to 4-ethylphenol (4EP), and bioengineered gut bacteria
to selectively produce 4EPS in mice. 4EPS entered the brain and was associated with changes
in region-specific activity and functional connectivity. Gene expression signatures revealed
altered oligodendrocyte function in the brain, and 4EPS impaired oligodendrocyte maturation
in mice as well as decreased oligodendrocyte-neuron interactions in
ex vivo
brain cultures. Mice
colonized with 4EP-producing bacteria exhibited reduced myelination of neuronal axons. Altered
myelination dynamics in the brain have been associated with behavioral outcomes
7
,
9
–
14
,
13
,
14
.
Accordingly, we observed that mice exposed to 4EPS displayed anxiety-like behaviors, and
pharmacologic treatments that promote oligodendrocyte differentiation prevented the behavioral
effects of 4EPS. These findings reveal that a gut-derived molecule influences complex behaviors
in mice via effects on oligodendrocyte function and myelin patterning in the brain.
A microbial biosynthetic pathway for 4EP
Previously, the metabolite 4EPS was measured at higher relative abundance in a mouse
model of atypical neurodevelopment, and systemic delivery of synthetic 4EPS to naïve
mice altered behavior in the open-field test
8
. We recently reported that 4EPS is elevated
in the plasma of individuals with autism spectrum disorder (ASD)
15
, and show here it
is increased in the blood of the CNTNAP2 model of ASD (Extended Data Fig. 1f). The
gut microbiome is predicted to harbor genes that convert tyrosine, the source of several
mammalian neurotransmitters, to 4-ethylphenol (4EP), which could then be sulfated
16
to
4EPS by the host (Fig. 1a). Consistent with this notion, germ-free (GF) mice devoid of a
microbiota contain virtually no detectable levels of 4EPS (Extended Data Fig. 1f)
8
. Some
rare bacterial species in the Firmicutes phylum produce 4EP using
p
-coumaric acid as a
substrate
17
,
18
, and precursors of
p
-coumaric acid include tyrosine or plant-based molecules
that can be metabolized by the gut microbiota to 4EP. Indeed, both a high-tyrosine fish-
based diet and a soy-based diet resulted in measurable 4EPS levels in conventionally
colonized mice (Extended Data Fig. 1g). By screening candidate gut bacterial isolates, we
discovered that
Bacteroides ovatus
produces
p
-coumaric acid from tyrosine (Fig. 1b). Using
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the basic alignment search tool (BLAST), we identified a tyrosine ammonia lyase in
B.
ovatus
(BACOVA_01194) that upon deletion, abrogated
p-
coumaric acid production (Fig.
1b).
We co-colonized GF mice with
B. ovatus
and
Lactobacillus plantarum,
the latter of which
can subsequently convert
p
-coumaric acid to 4EP
18
; however, the resulting 4EPS levels in
urine were low (Extended Data Fig. 1f, h). To improve the efficiency of 4EP production, we
carried out several rounds of strain engineering (see Methods). Briefly, an extra copy of the
first two genes in the pathway, BACOVA_01194 and the phenolic acid decarboxylase (
pad
),
were inserted into
B. ovatus
as a single, highly expressed operon (Fig. 1c). The engineered
B. ovatus
strain MFA05 robustly converts tyrosine to the intermediate, 4-vinylphenol
(Fig. 1c, Extended Data Fig. 1a-e), and in co-culture with
L. plantarum
, 4-vinylphenol is
quantitatively metabolized to 4EP (Fig. 1d). In contrast, when the
B. ovatus
∆1194 mutant
was co-cultured with
L. plantarum
, no 4EP was detected (Fig. 1d). We find homologs of
each gene in ~25 genomes of sequenced human gut microbes, indicating common pathways
may be intact in the human microbiome (Extended Data Fig. 1i).
Gut-derived 4EPS in the circulation and brain
We colonized separate groups of GF mice with either of the engineered strain pairs
represented in Fig. 1d, generating 4EP+ or 4EP- animals. As expected, 4EP was detected in
feces (Fig. 1e), and its host-sulfated derivative, 4EPS, was detected in the serum and urine
of 4EP+ colonized mice (Fig. 1f, Extended Data Fig. 1j). 4EP was undetectable in the serum
of 4EP+ mice, suggesting efficient sulfation to 4EPS (Extended Data Fig. 1j). Conversely,
4EP- mice do not have measurable 4EP or 4EPS levels (Fig. 1e-f, Extended Data Fig.
1j). 4EPS was detectable in brains of 4EP+ mice treated with probenecid, which inhibits
organic anion transporters that mediate efflux of small molecules through the blood brain
barrier, suggesting accumulation of 4EPS in the brain (Fig. 1g, Extended Data Fig. 1k-m).
We observed sulfation of 4EP to 4EPS by the sulfotransferase SULT1A1 and others during
in vitro
biochemical reactions (Extended Data Fig. 2a-b). SULT1A1 is found in intestinal,
liver, and brain tissues of mice (Extended Data Fig. 2c-d), though the site(s) of 4EP sulfation
remains unknown.
4EP and 4EPS are phenolic molecules that may have toxic or inflammatory properties
19
.
However, we observed no differences between 4EP+ and 4EP- groups in body weight or
ambulatory activity (i.e., locomotion) (Extended Data Fig. 2f-g). No evidence of intestinal
dysfunction was detected in 4EP+ mice when assessing epithelial permeability (Extended
Data Fig. 2h), fecal output (Extended Data Fig. 2i), or gross histopathology (Extended Data
Fig. 2j). Bacterial colonization levels and ultrastructural localization of bacteria were similar
between groups of mice (Extended Data Fig. 2k-m). We did not observe pro-inflammatory
cytokine responses in colonic tissue or serum (Extended Data Fig. 3a-b), and only modest
changes in peripheral immune cell proportions (Extended Data Fig. 3c-d). Cytokine levels
trended toward an anti-inflammatory profile in the brain with no signatures of microglial
activation in 4EP+ mice (Extended Data Fig. 3e-g). Collectively, these studies establish a
simplified animal model that reproduces the natural route of exposure to a gut microbial
metabolite associated with altered behaviors (Extended Data Fig 2e).
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4EPS-dependent brain activity patterns
While 4EP or 4EPS --designated here as 4EP(S)-- may have effects on various organs, we
focused our study on the brain. Initially, to capture brain-wide differences between 4EP+
and 4EP- mice, we performed functional ultrasound imaging (fUSi), an
in vivo
method
that measures resting state cerebral blood volume variation to assess functional connectivity
(Extended Data Fig. 4a). We observed altered (mostly increased) correlation of signaling
patterns within 4EP+ mice compared to 4EP- mice (Fig. 2a). These changes were primarily
observed in subregions of the hippocampus, thalamus, amygdala, hypothalamus, piriform,
and cortex (Fig. 2b, Extended Data Fig. 4b), indicating that elevated 4EPS is associated with
aberrant functional connectivity between various brain regions in mice.
To compare neural activity across the brain, we mapped glucose uptake where a systemically
injected radiolabeled tracer [(
14
C]-2-deoxyglucose, 2DG) is rapidly incorporated into active
brain regions. Changes in brain activity were evaluated by autoradiography of brain sections
comparing 4EP+ to 4EP- mice. 4EP(S) was associated with increased glucose uptake
in subregions of the hypothalamus (anterior area; lateral; and paraventricular nucleus),
amygdala (anterior, basolateral, central, cortical), and the bed nucleus of the stria terminalis
(BNST), as well as in the paraventricular nucleus of the thalamus (PVT) (Fig. 2c,d). We
also mapped uptake during a behavioral task (open-field exploration) where we observed
overlap in increased activity in some regions (amygdala, hypothalamus, BNST, PVT), with
differences in the spatial extent of these changes between stimuli conditions (Extended
Data Fig. 4c-e). The regions highlighted by this analysis are important for a range of
functions, including mediating appropriate responses to innate and learned fear stimuli
20
–
23
and anxiety responses
24
,
25
. We conclude that gut exposure to 4EP in mice results in altered
functional connectivity and activity in multiple brain regions, including several associated
with the limbic system.
Altered oligodendrocyte maturation
To resolve molecular effects of 4EP(S) on the brain, we performed mRNA sequencing
(QuantSeq) of six brain regions from 4EP+ and 4EP- mice, including the PVT,
basolateral amygdala, hypothalamus, BNST, medial prefrontal cortex (mPFC), and ventral
hippocampus, resulting in tight clustering of transcriptomic profiles by brain region
(Extended Data Fig. 5a). 4EP(S) predominantly affected global gene expression in the
PVT, and the BNST and basolateral amygdala to a lesser extent (Fig. 3a, Extended Data
Fig. 5b, e). Differentially expressed genes were aggregated into functional categories
using annotated Gene Ontology (GO) terms, disclosing that the Notch signaling pathway
was elevated in the PVT of 4EP+ mice, while GO terms associated with dendrite
and neuronal projection development were decreased (Extended Data Fig. 5c-d). Cell-
specific enrichment analysis revealed decreased expression of genes specific to neurons,
newly formed oligodendrocytes, and mature oligodendrocytes in the PVT of 4EP+ mice
compared to 4EP- mice (Fig. 3b), suggesting that a potential decrease in development,
abundance, and/or activity of these cell types is associated with exposure to 4EP(S).
Increased proliferation of immature oligodendrocytes and decreased differentiation into
mature oligodendrocytes has been associated with elevated Notch signaling
26
,
27
. Mature
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oligodendrocytes insulate neuronal projections with myelin, a fatty sheath that promotes
conduction of action potentials along axons
28
. Accordingly, many genes that are hallmarks
of mature oligodendrocytes such as the myelin oligodendrocyte glycoprotein (
Mog
) and
Opalin
genes were downregulated in the PVT of 4EP+ mice, while several genes associated
with non-myelinating, oligodendrocyte progenitor cells (OPCs) were elevated (Fig. 3c, Ext.
Data Fig. 5f, Supplementary Information). Using seed analysis of the 2DG-uptake data
29
,
we observed fewer significant correlations between the PVT and the rest of the brain in the
4EP+ group (168,042 voxels) compared to the 4EP- group (271,392 voxels), an effect that is
largely driven by the reduced number of positive correlations in the 4EP+ compared to the
4EP- group (61,572 vs 141,493 voxels) (Fig. 3d, Extended Data Fig. 5g).
We tested the hypothesis that 4EP(S) impacts oligodendrocyte maturation. Immunostaining
in the PVT revealed increased expression of neural/glial antigen 2 (NG2) in 4EP+ mice,
indicative of immature, oligodendrocyte precursor cells, along with decreased levels of
a mature oligodendrocyte marker stained by a CC1 antibody (Fig. 3e-f, Extended Data
Fig. 5h-o). Analysis by flow cytometry and Western blot corroborated a skewing toward
immature oligodendrocytes in 4EP+ mice (Fig. 3g, h, Extended Data Fig. 6a-c). Levels of
NeuN, a pan-neuronal marker, and OLIG2, a pan-oligodendrocyte marker, were unchanged
(Extended Data Fig. 5k-n), indicating effects on oligodendrocyte maturation by 4EP(S)
rather than a change in the total number of cells within this lineage. These data suggest that
4EP(S) exposure leads to reduced oligodendrocyte maturation.
Similar phenotypes were observed in organotypic brain slices cultured in the presence
of 4EPS. 4EPS-treated
ex vivo
brain tissue showed increased levels of the early
oligodendrocyte marker NG2 relative to the mature marker CC1 (Fig. 3i, Extended Data
Fig. 6d), and reduced colocalization of myelin with neuronal axons (Fig. 3j-k, Extended
Data Fig. 6e). Further, functional markers of mature oligodendrocytes (MOG and myelin
basic protein, MBP), were lower in 4EPS-treated samples, while transcription of the gene for
NG2 (
Cspg4
) was increased (Fig. 3l, Extended Data Fig. 6f-g). While 4EPS enters the brain
and has direct effects on brain tissue, we cannot exclude peripheral influences.
Reduced neuronal myelination in 4EP+ mice
We employed electron microscope tomography (ET) to examine the ultrastructure of myelin
in the dense and organized axonal tracts of the corpus callosum to facilitate myelin
quantification. We observed a striking increase in the ratio of unmyelinated to myelinated
axons in the brains of 4EP+ mice compared to 4EP- mice (Fig. 4a, d), and a decrease
in normalized (indicated by an increased g-ratio) and actual myelin thickness in 4EP+
mice (Fig. 4b-d, Extended Data Fig. 7a-e). Thus, consistent with the decrease in mature
oligodendrocytes, 4EP(S) exposure reduces myelination frequency and efficiency in the
brain.
Diffusion tensor imaging (DTI), a magnetic resonance imaging modality that assesses
diffusion along myelinated tracts in the brain, was used to investigate the structural
connectivity of myelin between the PVT and the rest of the brain. We found lower fractional
anisotropy (FA), an indication of more dispersive rather than linear/restricted diffusion,
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in 4EP+ compared to 4EP- mice (Fig. 4e, Extended Data Fig. 7f). A similar defect in
myelination was observed in whole brains and trended in the corpus callosum (Extended
Data Fig. 7g-i). The significance of altered myelin dynamics and brain connectivity is an
emerging concept in behavioral neuroscience
10
–
12
,
30
,
31
.
4EP(S) increases anxiety-like behavior
Our previous study
8
and the 4EP(S)-dependent changes in the limbic system observed
here prompted us to investigate whether 4EP production in the gut can modulate complex
behaviors in mice. 4EP(S) promoted robust anxiety-like behavior in several testing
paradigms: 1) the elevated plus maze (EPM) where 4EP+ mice spend less time in the
terminus of the open arms, 2) open-field exploration where mice ventured less into the more
exposed zone of the arena, and 3) the light/dark box where 4EP+ mice spent more time in
the dark (Fig. 4f, g, Extended Data Fig. 8a-c). 4EP+ mice also displayed increased marble
burying, reflecting features of anxiety and/or stereotypic behaviors, but no increase in
self-grooming (Fig. 4h, Extended Data Fig. 8d). Beyond anxiety-like behaviors, 4EP+ mice
exhibited modestly altered social communication with increased anogenital sniffing in the
direct social interaction assay (Extended Data Fig. 8e). In the adult ultrasonic vocalization
(USV) test, male 4EP+ mice emit significantly fewer auditory communications to a novel
age-matched female (Extended Data Fig. 8f). Interestingly, there were no significant
differences in cognition or motor function between groups (via novel object recognition,
Y-maze alternation, beam traversal, pole descent, or wire hang tests) (Extended Data Fig.
8g-k), further suggesting the effects of 4EP(S) are selective for emotional behaviors. Our
reductionist model system employs an artificial microbiome to study the effects of a gut
microbial metabolite (Extended Data Fig. 8l). Importantly however, oral administration
of 4EP(S) to elevate levels in conventionally colonized mice also increased anxiety-like
behaviors and reduced oligodendrocyte maturation (Extended Data Fig. 9a-j). 4EP- mice
behaved similarly to conventionally colonized controls (Extended Data Fig. 9k,l), indicating
the behavioral effects of 4EP(S) are not specific to gnotobiotic mice.
Finally, we sought to determine whether the 4EP(S)-mediated effects on oligodendrocytes
contribute to altered behaviors. Administration of clemastine fumarate, a drug that promotes
oligodendrocyte maturation
32
, increased mature oligodendrocyte ratios in 4EP+ mice as
measured by CC1 and NG2 staining of brain sections (Fig. 4i,j, Extended Data Fig. 10a).
Notably, enhancing maturation of oligodendrocytes prevented behavioral changes in 4EP+
mice, including alterations in EPM, open field, and the marble burying tests (Fig. 4k-m,
Extended Data Fig. 10c,e,f). We observed similar improvements in anxiety-like behaviors
with another myelination-inducing drug, miconazole (Extended Data Fig. 10b,d, g-i). We
conclude that 4EP(S) impacts anxiety-like behaviors in mice in a manner that includes
effects on oligodendrocyte maturation.
Discussion
Herein, we discovered a biosynthetic pathway for production of the gut microbial metabolite
4EP. While this pathway can utilize tyrosine as a precursor, we show that other dietary
sources can also be metabolized by the gut microbiota into 4EP, and expect that in humans,
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diverse dietary and microbial community structures may impact circulating metabolite
levels. Additionally, we show that 4EP is sulfated and 4EPS enters the brains of mice, is
associated with altered activation and connectivity of specific brain regions, and disrupts the
maturation of oligodendrocytes and myelination patterns in the brain. Environmental cues
are known to have regional effects on oligodendrocyte-neural interactions, influencing brain
circuits that govern particular behaviors
6
,
7
. While other brain regions are likely involved in
the 4EP(S) response, as we detect broad changes to activation patterns, the PVT receives
sensory and cognitive input, integrates these cues locally, and exports finely tuned signals
to cortical and subcortical areas resulting in applied behavioral responses
33
. Indeed, we
show gut exposure to 4EP alters several emotional, but not non-emotional, behaviors in
mice. Future work will focus on uncovering how 4EPS leads to changes in oligodendrocyte
maturation and myelination, defining brain regions that are causally affected, and exploring
how myelination changes impact behavior. Our data do not resolve if 4EPS is the
neuroactive metabolite, versus 4EP or unknown breakdown products. Identification of gut-
derived microbial metabolites that enter the brain and affect brain activity defines a novel
environmental influencer of anxiety-like behaviors.
Gut bacteria can synthesize classical neurotransmitters such as dopamine, norepinephrine,
serotonin and gamma-aminobutyric acid, and the production of novel classes of neuroactive
metabolites by the microbiome has been postulated
34
. Molecules with phenolic structures
similar to 4EP(S) are dysregulated in several preclinical models of behavior
8
,
35
,
36
as
well as in certain neuropsychiatric disorders
37
–
41
. Intriguingly, a metabolite of tyrosine
closely related to 4EP,
p
-cresol, has been suggested to influence oligodendrocyte function
and affect social and depression-like behaviors in mice
6
,
42
. Increased 4EPS is associated
with abnormal repetitive behavior in non-human primates
36
, and plasma levels of 4EPS
are significantly increased in a subset of individuals with ASD
15
. Additionally, relative
abundances of several related metabolites such as 2-ethylphenylsulfate, 4-allylphenyl sulfate,
and 4-methylbenzenesulfonate are altered in this same cohort
15
. We propose the hypothesis
that 4EP(S) represents the archetypical example of a neuroactive microbial molecule that
impacts brain activity and complex behaviors in animals, conceptually akin to mammalian
neurotransmitters that regulate nervous system function.
Materials and Methods
Bacterial strains and culture conditions
All bacterial strains and plasmids used in this study are shown in Supplementary
Information Tables 1 and 2.
B. ovatus
was cultured in brain heart infusion (BHI) agar
medium supplemented with 10% horse blood, TYG (tryptone-yeast extract-glucose) broth
or minimal medium (MM) at 37 ̊C in an anaerobic chamber from Coy Laboratories
44
,
45
.
Tyrosine was supplied as a substrate during investigation of 4EP production.
L. plantarum
was cultured anaerobically in MRS media (BD) at 37 ̊C.
Escherichia coli
strains were
cultured aerobically in LB broth. When appropriate, the growth medium was supplemented
with 100 μg/ml carbenicillin, 25 μg/ml erythromycin, 2 μg/ml tetracycline, 200 μg/ml
gentamicin and 200μg/ml 5-fluoro-2’-deoxyuridine (FUdR).
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Disruption of BACOVA_01194 in
B. ovatus
Using NCBI BLAST alignment tools, we predicted that BACOVA_01194 was an ammonia
lyase that could metabolize tyrosine. For all PCR amplification steps we used PrimeSTAR
Max DNA polymerase (Takara Bio, Mountain View, CA) according to the manufacturer’s
instructions. Primer sequences are shown in Extended Data Table 3. We used a previously
reported double-crossover recombination method to construct
B. ovatus
Δ1194
46
. Briefly,
~1 kb DNA fragments corresponding to the upstream and downstream regions of the
target gene were PCR amplified and then digested with restriction endonucleases. The
digested fragments were then ligated into the suicide plasmid pExchange-
tdk
using T4 DNA
ligase (New England Biolabs, Ipswich, MA). The resulting plasmid was transformed into
Escherichia coli
S17–1
λ
pir
by electroporation and transformants were confirmed by PCR
amplification of the junction regions. An
E. coli
clone harboring the plasmid was cultivated
and plasmid DNA was isolated, purified, and verified by DNA sequencing.
For conjugation into
B. ovatus, B. ovatus ∆tdk
and
E. coli
S17–1
λ
pir
harboring the plasmid
were cultivated and the cells were harvested by centrifugation. The cell pellets were washed
with PBS to remove residual antibiotics and combined in TYG medium. The suspension was
plated on BHI-blood agar medium without any antibiotics and cultivated aerobically at 37
̊C for 1 day. The bacterial biomass was recovered by scraping and re-suspended in TYG
medium. The suspension was then plated on BHI-blood agar medium supplemented with
erythromycin and gentamicin and single-crossover integrants were selected. These strains
cultured in TYG medium overnight and plated on BHI-blood agar medium supplemented
with FUdR. The deletion mutant was screened by PCR amplification and verified by DNA
sequencing.
B. ovatus
strain engineering
When a culture of
L. plantarum
was supplemented with 4-vinylphenol, it was converted
quantitatively to 4-ethylphenol; in contrast, when the culture was supplemented with
p
-coumarate, the conversion to 4-vinylphenol was slow. This suggested that the
decarboxylation step might be rate-limiting for 4-ethylphenol biosynthesis in binary culture.
To address this challenge, we introduced the
pad
gene from
L. plantarum
into
B. ovatus
in order to enable
B. ovatus
to produce 4-vinylphenol directly from tyrosine. However, the
resulting strain did not produce 4-vinylphenol. We next introduced a second copy of the
BACOVA_01194 gene into
B. ovatus
along with
pad
in case the intermediate p-coumarate
was limiting, but this strain failed to produce 4-vinylphenol. To address the possibility that
the
pad
gene from
L. plantarum
does not function robustly in
B. ovatus
,
pad
from
Bacillus
subtilis
was introduced into
B. ovatus
; the engineered strain produced 4-vinylphenol robustly
from tyrosine. To boost the level of 4-vinylphenol, a second copy of BACOVA_01194 was
introduced into
B. ovatus
along with
pad
from
B. subtilis
using a second integration vector
with a different antibiotic marker. This strain produced 4-vinylphenol more robustly. We
then combined the second copy of BACOVA_01194 and
pad
into a single artificial operon
driven by a strong phage promoter and introduced this construct into
B. ovatus
. Interestingly,
this strain produced a much higher level of 4-vinylphenol and it was used throughout
the manuscript for co-cultivation and colonization. Vector maps and sequences of primers
used for
B. ovatus
engineering are shown in Extended Data Fig. 1 and Supplementary
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Information Table 3, respectively. The artificial operon was constructed in the following
way: A phage promoter along with 5’ and 3’ flanking sequences from the target vector,
pNBU2, and one of the target genes was amplified by PCR. The target gene with 5’ and 3’
flanking sequences containing sequences of the phage promoter and the pNBU2 vector were
also amplified. These two PCR fragments were assembled by overlap PCR. The BO_01194
and
pad
genes were each assembled by one more round of overlap PCR. The fragment
containing the target gene and the phage promoter was cloned into the fragment of pNBU2
vector using Gibson Assembly. The assembled plasmids were transformed into
Escherichia
coli
S17–1
λ
pir
competent cells by electroporation and transformants were confirmed by
PCR amplification of the junction regions. The positive clone harboring assembled plasmid
was cultivated and the plasmid was isolated, purified, and confirmed by DNA sequencing.
To conjugate the plasmid into
B. ovatus, B. ovatus ∆tdk
and
E. coli
S17–1
λ
pir
harboring
the plasmid were cultivated and the cells were harvested. The cell pellets were washed with
PBS to remove residual antibiotics and combined them in TYG medium. The suspension
was plated on BHI-blood agar medium without any antibiotics and grown aerobically at 37
̊C for 1 day. The bacterial biomass was recovered by scraping and re-suspended in TYG
medium. The suspension was then plated on BHI-blood agar medium supplemented with
erythromycin and gentamicin and antibiotic resistant strains were selected followed by PCR
amplification of the junction regions.
Extraction of bacterial culture metabolites
For metabolite analysis,
B. ovatus
was cultured in TYG medium overnight and the cells
were harvested and washed with MM. The cell pellet was re-suspended in MM containing
0.5 mg/ml tyrosine to a density of OD
600
= 1.0 and incubated anaerobically for 1 day. The
culture was extracted with acetone (20% v/v) and centrifuged. The supernatant was analyzed
by LC/MS as described below. For LC-MS analysis of co-culture experiments,
B. ovatus
and
L. plantarum
were cultivated anaerobically in TYG and MRS, respectively overnight and
the cells were harvested and washed with MM. The cell pellet was re-suspended in MM
containing 0.5 mg/ml tyrosine to a density of OD
600
= 1.0 and combined in the same culture
tube. After 1-day incubation, samples for HPLC analysis were prepared as described above.
LC/MS analysis of bacterial culture metabolites
Metabolite extracts were analyzed using an Agilent 1260 LC system coupled to an Agilent
6120 quadrupole mass spectrometer with a 3 μm, 4.6 × 75 mm Unison UK-C18 column
(Imtakt, Portland, OR). Water with 10mM ammonium acetate and 0.1% formic acid (A)
and acetonitrile with 0.1% formic acid (B) was used as the mobile phase at a flow rate
of 1.0 ml/min with the following 20 min gradient: 0–5 min, 0% B; 5–17 min, 0–90% B;
17–20 min, 95% B. P-coumaric acid and 4-VP were detected at 280nm and these retention
times were ~10.3 min and ~12.6 min, respectively. Retention time of 4-EP was ~7.6 min.
Standards of p-coumaric acid and 4-ethyl phenol were purchased (Sigma, St. Louis, MO).
Gene sequence alignment
Sequences of the genes used for strain engineering were aligned against the reference
genomes in the WoL database, which were pre-annotated using UniRef release 2019_07.
The annotation files are publicly available at:
https://biocore.github.io/wol/download
. The
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alignment used DIAMOND v0.9.25 with all default parameters (`diamond blastx-d/path/to/
db-qinput.fa-o output.txt`). The output files were then processed using Woltka (
https://
github.com/qiyunzhu/woltka
), which generated the taxonomic and functional (UniRef)
profile of the three genes against the entire WoL genome catalog.
Mouse Husbandry
All animal husbandry and experiments were approved by the Caltech Institutional Animal
Care and Use Committee. Throughout the study, animals were maintained in autoclaved
microisolator cages with autoclaved bedding (Aspen Chip Bedding, Northeastern Products
Corp, Warrensburg, NY), water, and chow (Laboratory Autoclavable Rodent Diet - 5010,
LabDiet; St. Louis, MO, USA). Diets used in Extended Data Fig. 1g were purchased from
Envigo Teklad (Madison, WI), carbohydrate, and fat content. The soy protein has ~300ppm
total isoflavones. The tyrosine levels are 4.6g/kg in soy diet and 17.1g/kg in fish diet. 8-wk,
SPF C57BL/6J male mice (Jackson Laboratory, Bar Harbor, ME), were provided these
special diets for 2 weeks ad libitum prior to urine collection for metabolite analysis as
described below.
Mice were maintained at an ambient temperature of 71–75F, 30% - 70% humidity, at a cycle
of 13 hours light & 11 hours dark.
Experimental Design of Mouse Experiments
Germ-free (GF) C57BL/6J male weanlings (3 weeks of age) from the Mazmanian laboratory
colony were colonized by gavage of 100ul of 1:1 mixture of 10
9
CFU/ml
B. ovatus
(+/−
4EP pathway genes) and wild type
L. plantarum
. This process was continually performed,
yielding a steady schedule of cohorts for continued study throughout this work. Size of
animal groups was determined by the largest number of pups that could be born within the
space of the germ-free isolators. Weekly tests to confirm gnotobiotic status were performed
and 4EPS levels were confirmed in urine regularly. All experiments in the study were
repeated on at least two independent cohorts. Preliminary behavioral analysis identified a
stronger phenotype in males (Extended Data Fig. 10j-l), so they were used for the remainder
of the study in effort to limit animal use.
16S sequencing was performed by Laragen (Culver City, CA) and analysis was done with
a 16S V6 library. Paired-end fastq files were processing using Qiime2
47
. Briefly, sequences
were quality control processed with dada2
48
, truncating reads to 150 bp. Taxonomic
classification was then conducted using the greengenes database. Analysis was conducted on
samples rarefied to 24,000 reads.
In the conventionally colonized (SPF) experiment, male weanlings (3 weeks of age) were
provided 250mM 4EP and 4EPS in drinking water (or vehicle). In these experiments, oral
administration continued until endpoint. 4EPS was synthesized as previously described
8
,
and 4EP was purchased from Sigma, (St. Louis, MO). When appropriate, beginning at 4–5
weeks of age, sterile clemastine fumarate (0.03mg/ml) or DMSO vehicle (Sigma, St. Louis,
MO) was added to drinking water and water was changed every other day, or miconazole
solution (40mg/kg) or 3% DMSO vehicle was gavaged once daily. In all cases, behavior
testing started at 6 weeks of age.
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Extraction of urine, feces, serum and brain metabolites
Urine was collected by placing autoclaved aluminum foil under the mouse while briefly
scruffing it, and then pipetting the urine from the foil. The urine was diluted 5-fold with
aqueous acetonitrile (50% v/v) and centrifuged. The supernatant was analyzed by LC/MS
to detect 4EPS. 4EPS for a standard was prepared as previously described
8
. Ethyl acetate
was added to the supernatant to create a 1:1:1 mixture (v/v) of water, acetonitrile, and ethyl
acetate. After mixing and centrifugation, the organic layer was analyzed by GC/MS to detect
4EP.
Fecal pellets were collected by placing the mouse briefly into a sterile plastic beaker. A
5x volume of 50% acetonitrile/water was added and the pellets were homogenized by bead
beating, followed by centrifugation. The supernatant was analyzed by LC/MS to detect
4EPS. To detect 4EP, samples were extracted with ethyl acetate as described above.
Blood was collected by cardiac puncture followed by separation using Sarstedt Serum-Gel
microtubes (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s
specifications.
Brain tissue was collected by cardiac perfusion as described for immunohistochemistry
but with PBS instead of PFA, then dissected. A 2x volume of 50% acetonitrile/water was
added and the tissue was homogenized by bead beating, followed by centrifugation. The
supernatant was directly injected for analysis by LC/MS to detect 4EPS.
LC/MS analysis of metabolites from urine, serum, and brain
Samples were analyzed using an Agilent 1290 LC system coupled to an Agilent 6530 QTOF
with a 3 μm, 4.6 × 75 mm Unison UK-C18 column using the same method described above.
All data were collected in negative ion mode. 4EPS was detected as [M-H]- (calculated m/z
is 201.0227) and retention time was ~7.9 min. 4EPS prepared previously was used as a
standard
8
.
GC/MS analysis of metabolites from feces
Samples were analyzed with a split ratio of 10:1 using an Agilent 7890 GC coupled to an
Agilent 5977 MSD with a HP-5MS fused silica capillary column (30m x 250 μm x 0.25
μm). The injector temperature was set at 250 ̊C and high purity helium gas was used as
carrier at a constant flow rate of 1.0 ml/min. The column temperature was initially kept on
40 ̊C for 2 min, then increased to 100 ̊C at a rate of 40 ̊C/min, then went up to 105 ̊C at a
rate of 2 ̊C/min and then raised to 320 ̊C at a rate of 30 ̊C/min, held for 3 min, giving 16.367
min in total. Retention time of 4-EP (calculated m/z is 122.07) was ~7.6 min.
Creatinine measurement
Concentration of creatinine was measured using Colorimetric Creatinine Assay Kit (Abcam,
Cambridge, UK) according to the manufacturer’s instructions.
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Brain Levels of 4EPS
Probenecid (Invitrogen, Carlsbad, CA), an organic anion transporter (OAT) inhibitor that
works on OAT1 and OAT3 was injected intraperitoneally (i.p.) into mice (120mg/kg). After
1-hour, whole brains were removed, homogenized, and analyzed as described above by
LCMS. For SPF animals, 30 minutes after the probenecid injection, either an i.p. injection
of 100ul of 8mM 4EPS or an oral gavage of 160mg/kg 4EP (Sigma, St. Louis, MO) was
administered, then brains were harvested after 30 additional minutes or along 30-minute
time points. For mice colonized with 4EP- and 4EP+ strains, mice were perfused with PBS
before tissue collection to ensure any metabolite detected was not simply due to levels in the
blood.
qPCR
RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and cDNA was
transcribed using the iScript cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA).
qPCR was performed with the SYBR master mix (Thermo Fisher Scientific, Waltham, MA)
using the primers found in Supplementary Information Table 5.
In vitro assays of 4EP sulfation
Recombinant sulfotransferases (sult1b1, 1c2, 1e1, 2a1, and 1a1) and the Universal
Sulfotransferase Activity Kit (R&D Systems, Minneapolis, MN) were used according
to manufacturer’s recommendations. Following analysis by plate reader, samples were
analyzed by LCMS as described above to confirm that the sulfated product was indeed
4EPS. Cytosolic fractions from 50–200ug tissue containing endogenous sulfotransferases
were extracted and tested for SULT activity on 4EP as previously described
49
,
50
and
analyzed by LCMS as described above.
Intestinal Permeability
The FITC-dextran intestinal permeability assay was performed as described previously
51
on
4EP- and 4EP+ mice at nine weeks.
Hematoxylin and eosin (H&E) staining
Gut tissue was dissected immediately after sacrifice at nine weeks of age and fixed in neutral
10% formalin, paraffin embedded, sectioned, and stained with hematoxylin and eosin (H&E)
by Pacific Pathology, Inc, San Diego, CA.
Cytokine Analysis
Tissue samples collected at nine weeks of age were homogenized by bead beating in
lysing matrix D tubes (MP Biomedicals, Irvine, CA) in RIPA buffer (Millipore, Burlington,
MA) containing protease inhibitor tablets (Roche, Basel, Switzerland) followed by protein
quantification and normalization using the Pierce BCA Protein Assay Kit (Thermo Fisher
Scientific, Waltham, MA). Blood samples were collected by cardiac puncture followed
by serum isolation in clotting tubes (Sarstedt, Newton, NC). Using the Bio-Plex Pro
Mouse Cytokine 23-plex assay (Bio-Rad, Hercules, CA) according to manufacturer’s
recommendations, cytokine and chemokine levels were determined using a Bio-Plex 200
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Systems instrument. Undetected values were rare, and in those cases were imputed with the
lowest detected value from that cytokine. One-way ANOVA with multiple comparisons was
performed and significance is indicated with asterisks (*<0.05, **<0.01, ***<0.001).
Microglial enrichment for qPCR
Brain samples were collected and single cell suspensions were generated as described in
flow cytometry section below. Microglial-enriched populations were obtained using CD11b
microbeads (Miltenyi Biotec, San Diego, CA).
Animal protocol for functional ultrasound imaging (fUSi) session
14 mice were imaged in total: 7 mice for the 4EP- group and 7 for the 4EP+ group.
Images were acquired through intact skull and skin after hair removal using a commercial
depilatory cream (Nair™ , Church&Dwigth, USA) without any contrast agent injection.
During handling (shaving, positionning) mice were anaesthetized with isoflurane (2%)
administered in a mixture of 30% O
2
and 70% N
2
. During the resting state experiment,
animals were sedated using dexmedetomidine (Tocris Bioscience, Minneapolis, MN). A
bolus of 0.10 mg/kg was injected subcutaneously, and isoflurane was discontinued after 5
minutes. Mice were head fixed on a stereotaxic frame to minimize brain motion during
imaging. After a 90 min. imaging session, animals were euthanized by cervical dislocation.
Functional ultrasound imaging
Functional ultrasound imaging (fUSi) visualizes neural activity by mapping local changes in
cerebral blood volume (CBV). CBV variations are tightly linked to neuronal activity through
the neurovascular coupling
52
and are evaluated by calculated power doppler variations
in the brain
53
. fUSi was performed transcranially as described in
54
using a 15 MHz
ultrasonic ultralight probe prototype (15 MHz, 64 elements, 0.110 mm pitch, Vermon,
Tours, France) connected to a Verasonics Vantage ultrasound system (Verasonics Inc.,
Redmond, WA, USA) driven by custom Matlab® (MathWorks, USA) transmission scripts
(
https://github.com/brittanyneedham/Needham_Nature2022
)
55
. Each Power Doppler image
was obtained from the temporal integration of 220 compounded frames acquired at 500 Hz
frame rate, using 5 tilted plane waves separated by 3° (−6°, −3°, 0°, 3°, 6°) acquired at a
2500 Hz pulse repetition frequency (PRF). Power Doppler images were then repeated every
second (1Hz image framerate). Each block of 220 images was processed using a SVD clutter
filter
56
to separate tissue signal from blood signal to obtain a final Power Doppler image
exhibiting cerebral blood volume (CBV) in the whole imaging plane. Three coronal planes
per mice were scanned at a rate of 15min imaging time per plane, respectively Bregma
−0.9mm, Bregma −1.6mm, and Bregma −2mm.
Functional ultrasound data processing and statistics
Power Doppler data were collected continuously during the imaging session and
connectivity process was applied afterwards. We followed the functional connectivity
process on fUSi data described in Osmanski, et. al
57
. For each coronal plane, for each
acquisition, and each mouse: first, a low-pass filter (cutting-frequency: 0.2 Hz) was
performed on the Power Doppler temporal signals for each individual pixel of the image to
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remove high frequency signals while preserving the resting-state frequency band. The signal
was then detrended with a polynomial fit of order 4 to remove low frequencies which could
bias the correlation value. Finally, the normal score of the temporal filtered signals were
calculated to make possible correlation calculation. In order to build functional connectivity
matrices, we determined within each coronal plane regions of interest (ROIs) defined from
the Paxinos Atlas
43
(See Extended Data Fig. 2a for ROIs mapping in each plane). The
Pearson correlation of the filtered signals of each pair of ROIs’ within a same plane were
then calculated and the corresponding correlation values were stored in the cells where
regions intersect in the connectivity matrix. Fig.2 shows the mean connectivity matrices
from each coronal plane (Bregma-0.9mm, Bregma-1.6mm and Bregma-2mm) of each
studied group (4EP- and 4EP+). Within the same coronal plane, cells of the connectivity
matrices from the 4EP- and 4EP+ groups were statistically analyzed individually using a
paired t-test. Multiple comparison correction was ensured with a Bonferroni correction
58
.
Region pairs that showed significant differences between groups are shown in Fig. 2c.
ROIs distribution graphs of each coronal plane are provided.
Coronal plane B-0.9mm: ROIs
#1 to #8 are located in the left cortex, ROI#9 is the left hippocampus, ROIs#11 to #21 are
located in the thalamus, ROI#22 is the right hippocampus, ROIs#23 to #30 are located in the
right cortex and finally ROIs#31 to #48 are subthalamic regions.
Coronal plane B-1.6mm:
ROIs #1 to #20 are located in the cortex, ROIs#21 #22 are the left and right hippocampi,
ROIs#23 to #38 are located in the thalamus and ROIs#39 to #42 are subthalamic regions.
Coronal plane B-2mm: ROIs #1 to #9 are located in the left cortex, ROI#10 is the left
hippocampus, ROIs#11 to #22 are located in the thalamus, ROI#23 is the right hippocampus,
ROIs#24 to #32 are located in the right cortex and finally ROIs#33 to #50 are subthalamic
regions.
Autoradiography Brain Mapping
The autoradiographic 2DG uptake method is a well-established, time-tested approach to
functional brain mapping based on a tight coupling between neural activity and metabolism.
It is particularly suitable in awake, free-moving animals, and complements the fUSi
approach. Male mice colonized as described above were housed in pairs from weaning.
Mapping of cerebral glucose metabolism was performed as described previously
59
,
60
in four
groups: 4EP+/Home cage (
n
= 11), 4EP+/Open field (
n
= 11), 4EP-/Home cage (
n
= 10),
4EP-/Open field (
n
= 11). The experiment was performed in two cohorts with balanced
group assignment in each cohort. At 7 weeks mice were habituated to handling for 5 minutes
each day for 3 days prior to 2DG mapping. They were brought in their home cages to the
experimental suite 16 hours prior to mapping and were fasted of food overnight with water
ad libitum. A pair of mice from the same home cage were administered i.p. [
14
C]-2-deoxy-
D-glucose (Cat # MC355, radiochemical purity > 97%, specific activity 45 – 60 mCi/mmol,
Moravek Inc., Brea, CA, USA) at 0.3 μCi/g bodyweight in 0.5 ml normal saline. Animals
were placed back in their home cage for five minutes. One mouse was then placed into the
Open Field arena and allowed to explore the arena for 45 minutes to allow uptake of the
tracer, while the other remained in the home cage. At the end of exposure, following cervical
dislocation, brains were extracted and flash frozen in methylbutane over dry ice (~ −55
o
C)
and later serially sectioned into 20-μm slices in a cryostat at −20
o
C (Mikron HM550 OMP,
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Thermofisher Scientific, Waltham, MA, USA). Slices were heat dried on glass slides and
exposed to Kodak Biomax MR diagnostic film (Eastman Kodak, Rochester, NY, USA) for 3
days at room temperature. Autoradiographs were then digitized on an 8-bit gray scale using
a voltage stabilized light box (Northern Lights Illuminator, InterFocus Ltd., England) and a
Retiga 4000R charge-coupled device monochrome camera (Qimaging, Canada).
Relative regional cerebral glucose uptake (rCGU) was measured and analyzed on a whole-
brain basis using Statistical Parametric Mapping (SPM, version 5, Wellcome Centre for
Neuroimaging, University College London, London, UK) as previously described
59
,
61
.
Here briefly, each three-dimensional (3D) brain was reconstructed from 68 digitized
autoradiographs (voxel size: 40 × 140 × 40 μm) using TurboReg, an automated pixel-based
registration algorithm implemented in ImageJ (version 1.35,
http://rsbweb.nih.gov/ij/
). This
algorithm registered each section sequentially to the previous section using a nonwarping
geometric model that included rotations, rigid-body transformation, and nearest-neighbor
interpolation. One “artifact free” mouse brain was selected as reference, and all brains were
spatially normalized to the reference in SPM. Spatial normalization consisted of applying
a 12-parameter affine transformation followed by a nonlinear spatial normalization using
3D discrete cosine transforms. All normalized brains were then averaged to create a final
mouse brain template. Each original 3D-reconstructed brain was then spatially normalized
to the template. Normalized brains were smoothed with a Gaussian kernel (full width at half
maximum = 3x voxel dimension in the coronal plane). Voxels for each brain failing to reach
a specified threshold in optical density (70% of the mean voxel value) were masked out
to eliminate the background and ventricular spaces without masking gray or white matter.
Differences in the absolute amount of radiotracer uptake in the brain were normalized
in SPM for each animal by scaling the voxel optical densities such that the whole-brain
mean for each brain was the same (proportional scaling). For each condition (open field
and home cage exposure), one-tailed t-tests were performed voxel-by-voxel comparing
4EP+ and 4EP- animals. Threshold for significance was set at
P
< 0.05 at the voxel level
and an extent threshold of 200 contiguous voxels to eliminate false positive statistically
significant results. Color-coded functional overlays showing statistically significant changes
in rCGU were displayed over coronal sections of the template brain in MRIcro (version
1.40,
https://people.cas.sc.edu/rorden/mricro/mricro.html
). This combination reflected a
balanced approach to control both type I and type II errors. The minimum cluster criterion
was applied to avoid basing our results on significance at a single or small number of
suprathreshold voxels. Brain regions were identified according to a mouse brain atlas(
43
and
atlas.brain-map.org
).
A seed correlation approach was applied to assess 4EP-related differences in the functional
connectivity of the PVT. A structural region of interest (ROI) was hand drawn in MRIcro
over the template brain according to the mouse brain atlas for the PVT between bregma −1.0
and −1.6mm. Mean optical density of the seed ROI was extracted for each animal using
the MarsBaR toolbox for SPM (version 0.42,
http://marsbar.sourceforge.net
). Correlation
analysis was performed in SPM for each home cage group.
T
statistics were calculated using
a linear regression model with the seed value as the only covariate (regressor). Threshold
for significance of directional correlation was set at
p
< 0.05 (one-tailed
t
-test) at the
voxel level and an extent threshold of 200 contiguous voxels to serve as a proxy for
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multiple comparison correction, which is standard for the field. Regions showing statistically
significant correlations (positive or negative) in rCGU with the seed are considered
functionally connected with the seed. Color-coded functional overlays were displayed in
the template brain after 3D rendering in MRIcro to allow visual comparison of overall level
of functional connectivity.
Brain sample collection for immunohistochemistry
Mice were perfused via the cardiovascular system with PBS followed by 4%
paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA). Brains were removed and
post-fixed in 4% paraformaldehyde 1 day at 4°C. The brains were kept in PBS with 0.02%
sodium azide at 4°C until sectioning. For sectioning, the brains were embedded in 4%
UltraPure low melting point agarose (Thermo Fisher Scientific, Waltham, MA) and were
coronally sectioned by vibratome (VT1000S; Leica Microsystems, Wetzlar, Germany) at a
thickness of 50 μm. Brain sections of 50 μm were collected and stained every 0.15 mm.
The brain sections were stored as free-floating in PBS with 0.02% sodium azide at 4°C until
staining.
The free-floating sections were incubated with primary antibody in blocking solution (10%
horse serum, 0.3% triton X-100, and 0.02% sodium azide in PBS) overnight at room
temperature. The next day, sections were incubated with fluorescence-conjugated secondary
antibody for 1.5–2 hours at room temperature. Between each step and after secondary
antibody staining, sections were thoroughly washed with PBS or PBS with 0.1% triton-
X-100 at least three times for 10 minutes each. The stained free-floating sections were then
mounted onto the Superfrost Plus microscope slides (Fisher Scientific, Hampton, NH) in
PBS. Excess PBS from adhered sections were carefully removed. Slides were dried at room
temperature for 2–5 minutes. 150–200 μl of ProLong Diamond, anti-fade mountant with
DAPI (Thermo Fisher Scientific, Waltham, MA) was applied to the slides before placing the
coverslip. The slides were left to set overnight before imaging.
Primary antibodies used for imaging throughout and their dilutions were: mouse
anti-NeuN (1:1000; MAB377; Millipore Sigma, Burlington, MA), goat anti-Olig2
(1:500; AF2418; R&D Systems, Minneapolis, MN); mouse anti-CC1 (1:250; NB600–
1021; Novus Biologicals, Littleton, CO); rabbit anti-NG2 (1:300; AB5320; Millipore
Sigma, Burlington, MA); chicken anti-MBP (1:250; CH22112; Neuromics, Edina, MN);
mouse anti-neurofilament (1:250; 837802; Biolegend, San Diego, CA), rabbit anti-PLP
(ab183493, Abcam, Cambridge, UK). The fluorescent-conjugated secondary antibodies
were donkey anti-goat (1:1000; A-32814, A-21082, A11057; ThermoFisher Scientific,
Waltham, MA), donkey anti-rabbit (1:1000; A-21206, A-10042, A-31573; ThermoFisher
Scientific, Waltham, MA), and donkey anti-mouse (1:1000; A-21202, A-10037, A-31571;
ThermoFisher Scientific, Waltham, MA), and donkey anti-chicken (1:1000; A-11041;
A-11039; A-21449, Thermo Fisher Scientific, Waltham, MA).
Microscopic imaging and image analysis
Imaging was performed using the Zeiss LSM 800 inverted confocal laser scanning
microscope (Carl Zeiss, Oberkochen, Germany) with Zen software (Carl Zeiss, Oberkochen,
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Germany). Confocal images were obtained by Z-stacks covering the entire Z-axis range of
the sections. The interval for each focal plane was 2 μm intervals. The images were then
projected in the visualization plane with maximum intensity voxels (3D pixel) by maximum
intensity projection using Zen software. Positively stained cells were quantified using a
manual cell counter in ImageJ software (NIH). All images were minimally processed with
brightness and contrast adjustment. The adjustment was applied equally across the entire
image and consistent in the corresponding controls. Regions of interest were selected by a
segmented line based on the anatomical features of each region. The final number of positive
cells reported is averaged from 4 images.
Coordinates for imaging relative to Bregma (bilateral) were: BLA −1.06 to −2.06mm;
BNST: +0.38 to +0.26mm; PVN and AH: −0.70 to −0.94mm; mPFC: +1.10mm; PVT: −0.70
to −1.58mm; LHB and MBH: −1.06 to −1.34mm; SM: −0.46 to −1.34mm; ACA: 0.26mm;
CC: 0.26mm; LS: ; MS: 0.26mm; ME: −0.82mm.
QuantSeq
Following cervical dislocation, brains were extracted and placed in iced PBS for five
minutes. The brains were placed upside down into a brain matrix (SA-2175; Roboz,
Gaithersburg, MD) and sliced by single edge blades (Personna, Verona, VA). Brain slices
were left on the blades and placed on ice to maintain RNA integrity. Specific brain regions
were isolated by gross dissection or brain punches by using 1.0 mM Biopsy Punches
(Miltex, VWR, Radnor, PA) and were immediately frozen in RNAlater (Qiagen, Hilden,
Germany) until RNA collection following manufacturers recommendations using the Split
RNA extraction kit (Lexogen, Greenland, NH).
Coordinates for mPFC brain slices spanned from anterior to posterior (AP) +1.94 mm
to +1.10 mm relative to bregma. The gross anatomy of the mPFC was based on the
morphology of corpus callosum and the appearance of lateral septum. Coordinates for BNST
brain slices spanned from anterior to posterior (AP) +0.62 mm to +0.14 mm relative to
bregma (bilateral). The gross anatomy of the BNST was based on the features of caudate
putamen, lateral ventricle, and anterior commissure. Coordinates for PVT brain slices
spanned from anterior to posterior (AP) −0.94 mm to −1.58 mm relative to bregma. The
gross anatomy of the PVT was based on the appearance of dorsal hippocampus. Coordinates
for the hypothalamus spanned from anterior to posterior (AP) −0.58 mm to −2.92 mm
relative to bregma. The gross anatomy of the hypothalamus was based on the medioventral
part of the brain and covered numerous hypothalamic subregions. Coordinates for BLA
brain slices spanned from anterior to posterior (AP) −1.06 mm to −2.06 mm relative to
bregma (bilateral). The gross anatomy of the BLA was based on the terminal of external
capsule branches nearby the piriform cortex. Coordinates for vHPC brain slices spanned
from anterior to posterior (AP) −2.06 mm to −4.04 mm relative to bregma (bilateral). All
coordinates and diagrams were based on the Paxinos and Franklin atlas
43
.
Isolated brain tissue was immediately frozen in RNAlater (Qiagen, Hilden, Germany) until
RNA collection following manufacturers recommendations using the Split RNA extraction
kit (Lexogen, Greenland, NH). Quality control, library prep, and sequencing was performed
by the Penn State College of Medicine’s genome sciences facility as follows. The cDNA
Needham et al.
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