Slices were visualized using a 40× water im-
mersion objective (Zeiss Achroplan 440090;
NA 0.75; no aberration correction; working
distance 2.1 mm). We performed whole-cell
patch-clamp recordings from visually targeted
cells expressing both Td-Tomato and GFP in
the ARC. Patch pipettes were pulled from bo-
rosilicate glass (OD 1.5 mm, ID 0.86 mm; P-97
Flaming/Brown micropipette puller, Sutter
Instruments). These pipettes were filled with
a solution containing a mix of MDP or
MDPctrl (20.3mM), biocytin (0.15%, Sigma-
Aldrich), and potassium methanesulfonate–
based solution (126 mM K-methansesulfonate,
3.6mMKCl,3.6mMNaCl,9mMNa-Hepes,
10 mM Cs-EGTA, 0.3 mM GTP, and 4 mM
Mg-ATP, 280 to 290 mOsm, pH 7.3). The
solution passively diffused from the patch
pipette into the recorded cell after membrane
rupture in the whole-cell configuration. Re-
cordings were obtained via an Axon Multi-
clamp 700B. Data were acquired using Elphy
software (G. Sadoc, CNRS, Gif-sur-Yvette,
France). Recordings were performed in the
current-clamp mode. Constant current injec-
tion was used to hold membrane potential
near–70 mV. Hyperpolarizing current steps
(20 pA) were used to monitor input resist-
ance and incremental depolarizing current
steps (500 ms, 5 pA, up to 120 pA) were used
to elicit action potentials. The number of ac-
tion potentials elicited was analyzed for each
depolarizing step. This protocol was applied
just after membrane rupture and was repeated
after 10, 20, and 30 min of recording. Only
cells showing sustained trains of action po-
tentials at the initial time point were consi-
dered for further analysis. Membrane potentials
are corrected for a measured liquid junction
potential of 10 mV. The rheobase value was
the minimal depolarizing current pulse suffi-
cient to trigger an action potential. Record-
ings were analyzed with Elphy software. After
recording, brain slices were fixed overnight in
10% PFA and then washed three times in 1×
PBS. Slices were stained with anti-GFP as de-
scribed above and incubated with streptavi-
din (1:1000) in 1× PBS with 0.3% Triton X-100
for 30 min at room temperature. Slices were
then washed and mounted. Images were cap-
tured with 40× Zeiss lens (LD Plan-Neofluar
40×/0.6 Korr Ph 2M27) on a confocal laser-
scanning microscope (LSM 700, Zeiss).
Long-term antibiotic treatment
For mice injected with a Cre-expressing virus,
the antibiotic treatment was initiated 1 week
before the stereotaxic injections and lasted for
13 weeks. Mice received a cocktail of anti-
biotics in their drinking water, which con-
tained streptomycin (2.5 mg/ml), ampicillin
(1 mg/ml),vancomycin (0.5 mg/ml), and met-
ronidazole (0.5 mg/ml; all from Sigma-Aldrich).
The antibiotic-containing drinking water
was changed once a week and the bottles
were covered to avoid light exposure. Mouse
weights were measured and feces were col-
lected weekly.Fecal DNA extraction and bacterial qPCR
Fecal DNA was extracted using the FastDNA
spin kit (MP biomedical), following the recom-
mended protocol. Five nanograms of DNA were
used to perform the qPCR using SybrGreen
(BioRad). qPCR conditions and primers were
based on a previous publication ( 53 ). The
following sets of primers were used: pan-
bacteria: 5′-GCAGGCCTAACACATGCAAGTC-
3 ′(forward) and 5′-CTGCTGCCTCCCGTAGGA-
GT-3′(reverse); Actinobacteria: 5′-TACGGCCG-
CAAGGCT-3′(forward) and 5′-TCRTCCCCAC-
CTTCCTCCG-3′(reverse); Gammaproteobacteria:
5 ′-TCGTCAGCTCGTGTYGTGA-3′(forward) and
5 ′-CGTAAGGGCCATGATG-3′(reverse); Bacte-
roides: 5′-CRAACAGGATTAGATACCCT-3′(for-
ward) and 5′-GGTAAGGTTCCTCGCGTAT-3′
(reverse); and Firmicutes: 5′-TGAAACTYAAA-
GGAATIGACG-3′(forward) and 5′-ACCATGC-
ACCTGTC-3′(reverse).
The pan-bacteria Ct value for each mouse
was normalized to the mean Ct obtained from
feces collected just before ABX treatment was
begun. Bacterial group Ct values were normal-
ized to the Pan-bacteria Ct of the respective
mouse and day.Statistics
Statistical analysis was performed using com-
mercial analysis software (GraphPad Prism)
and with R 3.6.3 (www.R-project.org), withP≤
0.05 considered significant. Each experiment
was replicated at least twice. For the“nest-
test”analysis (Figs. 2G and 5L and fig. S4E),
due to the distribution of values associated
with the percentage of unrolled cotton, we
chose to compare discretized versions of the
corresponding distributions. Values were binned
as: class 1, 0 to 25%; class 2, 25 to 50%; class 3,
50 to 75%; class 4, 75 to 100%. The distribution
of events into these different classes was then
compared among the groups using a Fisher’s
exact test. The graphs show the data for each
mouse and not the class distribution for a
better appreciation of the raw values. For Fig.
5N analysis, the time is the driving force on
the two-way analysis of variance (ANOVA);
when looking into the contrast, one can ap-
preciate that the top driver of this force is the
control group (P= 0.0096), whereas the floxed
group is not significant (P= 0.085). For Fig. 1F
and fig. S3, B to E, the control group was not
considered for the statistical analysis because
this group did not receive any radiolabeled
PG-derivate. In supplementary figures, males
and females are shown in the same graph only
for a better graphical representation. However,
for statistical analysis, they were analyzed as
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