Effects of a probiotic add-on treatment on fronto-limbic brain structure, function, and perfusion in depression: Secondary neuroimaging findings of a randomized controlled trial

Abstract

Background
Probiotics are suggested to improve depressive symptoms via the microbiota-gut-brain axis. We have recently shown a beneficial clinical effect of probiotic supplementation in patients with depression. Their underlying neural mechanisms remain unknown.

Methods
A multimodal neuroimaging approach including diffusion tensor imaging, resting-state functional MRI, and arterial spin labeling was used to investigate the effects of a four-weeks probiotic supplementation on fronto-limbic brain structure, function, and perfusion and whether these effects were related to symptom changes.

Results
Thirty-two patients completed both imaging assessments (18 placebo and 14 probiotics group). Probiotics maintained mean diffusivity in the left uncinate fasciculus, stabilized it in the right uncinate fasciculus, and altered resting-state functional connectivity (rsFC) between limbic structures and the temporal pole to a cluster in the precuneus. Moreover, a cluster in the left superior parietal lobule showed altered rsFC to the subcallosal cortex, the left orbitofrontal cortex, and limbic structures after probiotics. In the probiotics group, structural and functional changes were partly related to decreases in depressive symptoms.

Limitations
This study has a rather small sample size. An additional follow-up MRI session would be interesting for seeing clearer changes in the relevant brain regions as clinical effects were strongest in the follow-up.

Conclusion
Probiotic supplementation is suggested to prevent neuronal degeneration along the uncinate fasciculus and alter fronto-limbic rsFC, effects that are partly related to the improvement of depressive symptoms. Elucidating the neuronal mechanisms underlying probiotics' clinical effects on depression provide potential targets for the development of more precise probiotic treatments.

Full text

Journal of Aective Disorders 324 (2023) 529–538
Available online 4 January 2023
0165-0327/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Research paper
Effects of a probiotic add-on treatment on fronto-limbic brain structure,
function, and perfusion in depression: Secondary neuroimaging ndings of
a randomized controlled trial
Gulnara Yamanbaeva
a
,
1
, Anna-Chiara Schaub
a
,
1
, Else Schneider
a
, Nina Schweinfurth
a
,
Cedric Kettelhack
a
, Jessica P.K. Doll
a
, Laura M¨
ahlmann
a
, Serge Brand
a
, Christoph Beglinger
b
,
Stefan Borgwardt
c
, Undine E. Lang
a
, Andr´
e Schmidt
a
,
*
a
University of Basel, Department of Psychiatry (UPK), Basel, Switzerland
b
Department of Research, St. Clara Hospital, Basel, Switzerland
c
Department of Psychiatry and Psychotherapy, University of Lübeck, Lübeck, Germany
ARTICLE INFO
Keywords:
Multi-strain probiotics
Depression
Microbiota-gut-brain axis
Uncinate fasciculus
Fronto-limbic network
Multimodal neuroimaging
ABSTRACT
Background: Probiotics are suggested to improve depressive symptoms via the microbiota-gut-brain axis. We have
recently shown a benecial clinical effect of probiotic supplementation in patients with depression. Their un-
derlying neural mechanisms remain unknown.
Methods: A multimodal neuroimaging approach including diffusion tensor imaging, resting-state functional MRI,
and arterial spin labeling was used to investigate the effects of a four-weeks probiotic supplementation on fronto-
limbic brain structure, function, and perfusion and whether these effects were related to symptom changes.
Results: Thirty-two patients completed both imaging assessments (18 placebo and 14 probiotics group). Pro-
biotics maintained mean diffusivity in the left uncinate fasciculus, stabilized it in the right uncinate fasciculus,
and altered resting-state functional connectivity (rsFC) between limbic structures and the temporal pole to a
cluster in the precuneus. Moreover, a cluster in the left superior parietal lobule showed altered rsFC to the
subcallosal cortex, the left orbitofrontal cortex, and limbic structures after probiotics. In the probiotics group,
structural and functional changes were partly related to decreases in depressive symptoms.
Limitations: This study has a rather small sample size. An additional follow-up MRI session would be interesting
for seeing clearer changes in the relevant brain regions as clinical effects were strongest in the follow-up.
Conclusion: Probiotic supplementation is suggested to prevent neuronal degeneration along the uncinate
fasciculus and alter fronto-limbic rsFC, effects that are partly related to the improvement of depressive symp-
toms. Elucidating the neural mechanisms underlying probiotics’ clinical effects on depression provide potential
targets for the development of more precise probiotic treatments.
1. Introduction
There is accumulating evidence showing the relevance of the gut
microbiota on the brain and its link to psychiatric disorders such as
depression (Cryan and Dinan, 2012). This connection is described as the
microbiota-gut-brain (MGB) axis and preclinical studies provided rst
insights on the specic underlying mechanisms in depression (Bravo
et al., 2011; Kelly et al., 2016; Zheng et al., 2016). Deciphering MGB
interactions may allow to detect novel biological targets for the
development of more efcient and tailored antidepressant treatments
(Cryan et al., 2019; Evrensel and Tarhan, 2021). Probiotics, which are
living bacteria that have benecial effects on the host (Markowiak and
´
Sli˙
zewska, 2017), are suggested to alleviate depressive symptoms by
modifying the MGB axis via the microbiome. Preclinical and clinical
studies found rst supporting results of benecial effects of probiotics on
depressive symptoms (Akkasheh et al., 2016; Goh et al., 2019; Huang
et al., 2016; Johnson et al., 2021; Ng et al., 2018; Yong et al., 2020). In a
recent study of our group, we found an antidepressant effect of a
* Corresponding author at: University of Basel, Department of Psychiatry (UPK), Wilhelm Klein-Strasse 27, 4002 Basel, Switzerland.
E-mail address: andre.schmidt@unibas.ch (A. Schmidt).
1
Shared rst authors.
Contents lists available at ScienceDirect
Journal of Affective Disorders
journal homepage: www.elsevier.com/locate/jad
https://doi.org/10.1016/j.jad.2022.12.142
Received 29 July 2022; Received in revised form 21 December 2022; Accepted 25 December 2022

Journal of Aective Disorders 324 (2023) 529–538
530
probiotic supplement in a sample of patients with current depressive
episodes (Schaub et al., 2022). Notably, this clinical improvement was
accompanied by increased abundance of the genus Lactobacillus.
Only few MRI studies have investigated effects of probiotics on brain
markers. A rst study, which could not nd any benecial behavioral
changes after a four-week probiotic supplementation in healthy partic-
ipants, showed reduced responses to emotional faces in a network
including the insula, periaqueductal gray and somatosensory cortex
(Tillisch et al., 2013). These results were supported by resting-state
functional connectivity (rsFC) analysis showing altered midbrain con-
nectivity after probiotics in healthy women. Further effects of probiotics
on brain functioning in healthy subjects were investigated in an analysis
on resting-state networks, showing decreased connectivity after a four-
week probiotic intervention in the default mode network, visual
network and a middle/superior frontal network compared to placebo
(Bagga et al., 2019). Conversely, increased rsFC was present in a salience
network including the cingulate cortex and precuneus. Notably, these
effects were not shown in brain structures measured with diffusion
tensor imaging (DTI), where only insignicant increases of fractional
anisotropy (FA) in the cingulum and the precuneus were observed.
Generally, on the neural level, patients with depression express ab-
normalities in both brain structure and function within a fronto-limbic
network (Drevets et al., 2008a; Fitzgerald et al., 2008; Liao et al.,
2013; van Velzen et al., 2020). One relevant associative white matter
tract in depression is the uncinate fasciculus (UF), which connects parts
of the limbic system including the temporal pole, subcallosal cortex,
cingulate gyrus, and amygdala (Catani et al., 2013; Morgane et al.,
2005) with inferior regions of the frontal lobe such as the orbitofrontal
cortex (OFC). Microstructural integrity abnormalities in the UF play a
role in the pathophysiology of depression (van Velzen et al., 2020) as
lower FA (LeWinn et al., 2014) and higher mean diffusivity (MD) (van
Velzen et al., 2020) have been reported along the UF in depressed pa-
tients compared to healthy controls. Relations between structural and
functional abnormalities in regions related to the UF in depression have
been described, showing reduced white matter integrity, expressed as
reduced FA along the UF, and increased task-based functional connec-
tivity between the subgenual anterior cingulate cortex (ACC) and the
medial-temporal lobe, changes that were related to the severity of
depressive symptoms (de Kwaasteniet et al., 2013). Also, increased ac-
tivity in limbic regions such as the amygdala, hippocampus, ventral
striatum (Anand et al., 2005; Mayberg et al., 1999), and the insula (Sliz
and Hayley, 2012), have been described compared to healthy controls as
well as decreased activity in various cortical areas, including sub-
divisions of the ACC, OFC, and dorsolateral prefrontal cortex (Mayberg
et al., 1999). However, divergent results on OFC connectivity depending
on its subregions have been reported, showing increased rsFC in the
lateral OFC connecting to the precuneus, posterior cingulate cortex and
angular gyrus in depressed patients, and decreased rsFC from the medial
OFC to the medial temporal lobe (Rolls et al., 2020). The subgenual ACC
has shown decreased gray matter volume and elevated metabolic ac-
tivity in patients with depression using positron emission tomography
(PET), which appeared to be reversed by antidepressant therapies
(Drevets et al., 2008b). Furthermore, regional cerebral blood ow (CBF)
was reduced in the ACC and bilateral parahippocampal areas and
increased in frontoparietal and striatal regions in depressed patients
using arterial spin labeling (ASL) (Vasic et al., 2015). In chronic
depressed patients, however, a hyperperfusion in the bilateral subgenual
ACC, left dorsomedial prefrontal cortex, left ACC and left subcortical
areas was found (Duhameau et al., 2010). Antidepressant treatment
with selective serotonin reuptake inhibitors and amesergide was related
to increased CBF in the left and mid ACC, left superior temporal gyrus,
and OFC as measured with single photon emission computed tomogra-
phy (SPECT) (Vlassenko et al., 2004). Another study found normaliza-
tions of reduced CBF in parieto/cerebellar regions after six-weeks
antidepressant therapy and decreased CBF in frontal regions after two
years (Kohn et al., 2008).
Here, we used a multimodal neuroimaging approach to investigate
whether probiotic supplementation over four weeks in depressed pa-
tients altered fronto-limbic network structure, function, and perfusion
by using DTI, resting-state functional MRI (fMRI), and ASL. Given that
probiotic supplementation ameliorated depressive symptoms in the
same sample (Schaub et al., 2022), we tested whether neural effects
were related to the benecial clinical response. We hypothesized that
probiotics would increase the structural integrity of the UF as expressed
by increasing FA and decreasing MD compared to placebo. We further
expected effects of probiotics on rsFC and CBF in regions along the UF,
specically increases in frontal areas and decreases in limbic regions
such as the amygdala. In addition, we predicted that the increased
structural integrity of the UF, as well as function and perfusion changes
in fronto-limbic regions are related to decreases of depressive symptoms.
2. Methods
This is a secondary analysis of a randomized controlled trial (RCT,
identier NCT02957591). Clinical and gut microbiota analyses have
previously been published (Schaub et al., 2022). Here we present nd-
ings of secondary outcomes such as DTI, resting-state fMRI and ASL.
2.1. Study sample
Patients with depressive episodes were recruited at the University
Hospital of Psychiatry (UPK), Basel, Switzerland and gave written
informed consent before participating in the study. The study was
approved by the local ethics committee (Ethikkommission Nordwest-
und Zentralschweiz). All patients were within inpatient care and
received treatment as usual (TAU) for depression. Detailed inclusion and
exclusion criteria are presented in the Supplementary material. The
initial sample size of 30 patients per group was calculated for the main
clinical outcome measure of this RCT as reported elsewhere (Schaub
et al., 2022). Out of 50 patients with baseline MRI data, 35 completed
the MRI assessment after the study intervention. As three patients were
<65 % compliant (Cramer and Rosenheck, 1998), they were excluded
from the imaging analysis resulting in a nal sample of 32 patients for
resting-state fMRI and ASL analyses (Supplementary Fig. 1). Analysis of
DTI data was conducted with 31 patients since one patient aborted the
MRI session before the DTI sequence. Medication was tracked over the
study period and summarized as dened daily dose (DDD) for antide-
pressants and antipsychotics separately. An overview of the study
sample characteristics is presented in Table 1.
Table 1
Study sample characteristics.
Probiotics
N =14
Placebo
N =18
Between-group
comparison
Demographics
Age (years), mean (SD) 38.36
(10.87)
36.83
(10.15)
t(27.08) =0.40, p
=.69
Sex, male/female 4/10 10/8
χ
2
(1) =1.36, p =
.24
Antidepressants (DDD), mean
(SD)
1.92 (1.45) 1.78
(0.93)
t(20.93) =0.31, p
=.76
Antipsychotics (DDD), mean
(SD)
0.13 (0.21) 0.20
(0.27)
W =120, p =.83
a
HAM-D
Baseline, sum score, mean (SD) 19.54
(4.89)
16.83
(4.37)
W =171.5, p =
.09
Change score: baseline -
postintervention, mean (SD)
10.68
(4.62)
8.33
(5.71)
W =163.5, p =
.16
Change score: baseline - follow-
up, mean (SD)
13.96
(4.46)
8.26
(4.40)
W =185.5, p <
.01**
Notes. DDD =dened daily dose, HAM-D =Hamilton Depression Rating Scale.
a
Non-parametric test since not normally distributed.
G. Yamanbaeva et al.

Journal of Aective Disorders 324 (2023) 529–538
531
2.2. Study design and intervention
In this double-blind RCT, subjects were allocated to two different
study groups and received either a probiotic supplementation or a pla-
cebo for 31 days in addition to TAU. The probiotics consisted of eight
different strains such as Streptococcus thermophilus NCIMB 30438, Bi-
dobacterium breve NCIMB 30441, Bidobacterium longum NCIMB 30435
(Re-classied as B. lactis), Bidobacterium infantis NCIMB 30436 (Re-
classied as B. lactis), Lactobacillus acidophilus NCIMB 30442, Lactoba-
cillus plantarum NCIMB 30437, Lactobacillus paracasei NCIMB 30439,
Lactobacillus delbrueckii subsp. Bulgaricus NCIMB 30440 (Re-classied as
L. helveticus). The daily dose contained 900 billion CFU/day that could
be mixed with any cold, non‑carbonated drink. In the control group,
participants took a placebo, which contained maltose but no bacteria
and was indistinguishable in color, shape, size, smell, and taste from the
probiotic supplement. Further details on the intervention are reported in
the Supplementary material. Patients received a standardized diet con-
taining stable amounts of bers and starch during inpatient setting.
Depressive symptoms were measured with the 17-items Hamilton
Depression Rating Scale (HAM-D) (Hamilton, 1967). Clinical and MRI
assessments were conducted before and after the four-weeks interven-
tion. Clinical data was also assessed at a four-week follow-up after the
end of the intervention.
2.3. MRI acquisition
DTI, resting-state fMRI and ASL data were acquired with a 3T
Siemens Magnetom Prisma Scanner before and after the probiotic
intervention The diffusion-weighted images were sampled with a RF-
pulsed gradient-spin-echo sequence during resting-state activity (inter-
leaved, FoV: 256 mm
2
, TR =7500 ms, TE =71 ms, slice thickness: 2
mm, 2 ×2 ×2 mm voxel size, EPI factor: 128, b-value =800 s/mm
2
including b0 images, i.e. a non-DWI’s, the “b =0 s/mm
2
”). The protocol
comprised 64 diffusion directions. RsFC was assessed with an 8.4 min
echo-planar T2* weighted imaging sequence (interleaved, FoV: 256
mm
2
, TR =3000 ms, TE =28 ms, 3 ×3 ×3 mm voxel size). Patients
were instructed to relax but stay awake and not to think of anything
specic during the acquisition. A structural sequence was used as
anatomical reference (MPRAGE, TR =2000 ms, TE =3.37 ms, 1 ×1 ×1
mm voxel size). ASL technique was used to estimate the CBF at resting-
state (Chen et al., 2015). Data were sampled with a RF-pulsed fast
gradient echo sequence (interleaved, FoV: 256 mm
2
, TR =3000 ms, TE
=12 ms, slice thickness: 4 mm, voxel size: 4 ×4 ×4 mm, EPI factor: 64,
20 transversal (axial) slices, PICORE Q2T perfusion mode, inversion
time: 1800 ms, bolus duration: 700 ms).
2.4. Structural properties analysis
DTI data was preprocessed using a standalone version of ExploreDTI
(Version 8.3, 2009) (Jeurissen et al., 2019; Tournier et al., 2011). In
brief, we rst performed the data reconstruction (Supplementary
methods). The preprocessing included subject motion and eddy-current-
induced distortions correction. Virtual dissection of the three-
dimensional UF tract was done as described by Wakana et al. (Wakana
et al., 2007). In brief, the ‘AND’ gates (Supplementary methods) were
located on a coronal slice at the position where the temporal and frontal
lobes are divided, allowing a selection of only those bers from the
temporal lobe, which projected to the frontal lobe. To omit ber pro-
jections that are not considered to belong to the UF, additional ‘NOT’
gates were placed when necessary (Fig. 1A). After preprocessing and
virtual dissection, FA and MD were calculated using ExploreDTI (Sup-
plementary methods).
2.4.1. Factorial ANOVA analysis
The UF tracts were dissected for all patients and parameters of the
ber tract structures were calculated. An entire set of the calculated tract
parameters (including FA and MD) was exported and further analyzed in
the software package STATISTICA (StatSoft, Inc., Tulsa, OK.: STATIS-
TICA, version 8). FA and MD parameters of the left and right UF at
baseline and post-intervention were entered in a factorial ANOVA
analysis to detect main effects of hemisphere, time and group, and their
interactions. In case of signicant main effects or interactions, post-hoc
Fig. 1. Uncinate fasciculus (UF) tract DTI trac-
tography. (A) Virtual dissection of UF. Green lines
at the coronal slice represents the ‘AND’ gates for
the virtual dissection. Virtually dissected left and
right UF shown in sagittal view. (B) Fractional
anisotropy (FA) of UF. The factorial ANOVA
analysis showed a signicant main effect of group
(F =7.433, p =.007) and hemisphere (F =7.288,
p =.008). (C) Mean diffusivity (MD) of UF. The
factorial ANOVA analysis showed a signicant
hemispheric effect (F =4.988, p =.027) and
time*group interaction (F =6.303, p =.013).
G. Yamanbaeva et al.

Journal of Aective Disorders 324 (2023) 529–538
532
testing using Tukey HSD test was performed.
2.5. Functional connectivity analysis
Resting-state fMRI data was analyzed using CONN toolbox (v19.c,
www.nitrc.org/projects/conn) (Whiteld-Gabrieli and Nieto-Castanon,
2012), which is an open-source Matlab/SPM-based software. A default
preprocessing pipeline for volume-based analyses was run including
functional realignment and unwrapping, slice-time correction, outlier
identication, segmentation and indirect normalization to MNI-space
and functional smoothing (6 mm kernel). Susceptibility distortion
correction using B0 eld maps with short and long real and imaginary
images was applied during the functional alignment and unwrapping
step. Data were denoised using linear regression of potential con-
founding effects in the BOLD signal (subject motion parameters, scrub-
bing (Power et al., 2014), noise components from white matter and
cerebrospinal areas and session effects). Furthermore, linear detrending
and temporal band-pass ltering (0.008–0.09 Hz) were applied.
2.5.1. Seed-to-voxel analysis
We investigated rsFC with a seed-to-voxel approach in which rsFC
maps are created for selected seeds. The study design with two groups
(probiotics and placebo) and two time points (baseline and post-
intervention) resulted in an 2 ×2 mixed ANOVA with interaction to
compare changes in rsFC between the two study groups. ROIs or rather
seeds-of-interest were chosen based on previous results linking the
medial temporal lobe and prefrontal regions to depression (Helm et al.,
2018). As ROIs, the subcallosal cortex (also referred to as subgenual
ACC), hippocampus and amygdala as well as further regions surround-
ing the UF such as the temporal pole, insula and OFC were included
(Supplementary Fig. 2). All ROIs were dened according to the Harvard-
Oxford cortical and subcortical structural atlas (Desikan et al., 2006).
First, all seeds were taken simultaneously in the model to avoid prob-
lems of multiple testing. Post-hoc tests were computed to determine
which selected seeds were responsible for the effects. Results were
corrected using false discovery rate (FDR) procedures and considered as
signicant with a p-FDR <0.05. When baseline differences in rsFC be-
tween groups occurred (two sample t-test), an ANCOVA with baseline
beta values as covariate, group as predictor and post-intervention beta
values as dependent variable was conducted in R. Beta values represent
Fisher-transformed correlations coefcients and were extracted from
CONN.
2.6. Blood perfusion analysis
ASL image preprocessing was performed using FSL6.0.0 (Smith et al.,
2004) and custom-written in Bash Unix shell (an interactive command
and scripting language that provides a command line user interface for
Unix-like operating systems, https://www.gnu.org/software/bash/)
routines, with help of FSL FEAT toolbox commands (Woolrich et al.,
2001). Preprocessing included motion correction, calibration of rst
volume against remaining volume as tag-control pairs, relative CBF
maps calculation, T1 structural scan reorientation and brain extraction
(Smith, 2002), calibrated volume image registration to T1, absolute CBF
calculation, thresholding, registration of thresholded images to T1 and
masking it, registration and normalization to MNI-space.
2.6.1. CBF ROI analysis
In line with the resting-state fMRI analysis, a CBF ROI analysis was
performed for the following regions: subcallosal cortex, hippocampus,
amygdala, insula, OFC and temporal pole (Supplementary Fig. 2). Ab-
solute CBF values were extracted and mean CBF for each patient was
calculated across each ROI separately (in ml/100 g/min). Mean CBF
estimates of each ROI were analyzed in STATISTICA (StatSoft, Inc.,
Tulsa, OK.: STATISTICA, version 8) using a factorial ANOVA analysis to
detect main effects of group, hemisphere and time, as well as their
interactions. In case of signicance, post-hoc Tukey HSD tests were
performed (Supplementary methods).
2.7. Correlation analysis
To investigate relationships between neuroimaging results and
depressive symptoms, parameters of all three imaging modalities were
linked to depressive symptoms using non-parametric Spearman rank
correlation. HAM-D change scores (baseline – post-intervention) were
correlated with FA and MD changes in the UF, beta values of seeds with
signicant interactions in rsFC and region-wise mean CBF estimates
within the probiotic and placebo group.
3. Results
3.1. Participants
Sample characteristics such as age, sex and medication were equally
distributed across the placebo and probiotic group (Table 1) and were
therefore not included in the analyses. Depressive symptoms decreased
in both study groups but changes were not statistically different between
the two groups from baseline to post-intervention. In the longer period
from the baseline to follow-up assessment, however, patients in the
probiotics group had a higher symptom decrease than patients in the
placebo group (Table 1).
3.2. Structural properties
3.2.1. Fractional anisotropy
The analysis of FA showed signicant main effects of group (F =
7.433, p =.007) and hemisphere (F =7.288, p =.008). Post-hoc testing
showed signicantly higher FA values in the probiotics (mean FA ±
SEM =0.428 ±0.003, p =.008) compared to the placebo group (0.417
±0.003) and signicantly higher FA in the left (0.427 ±0.003, p =
.007) than right UF (0.416 ±0.003) over both timepoints (Fig. 1B). No
signicant hemisphere*group (F =0.326, p =.569), time*hemisphere
(F =0.433, p =.512), time*group (F =1.324, p =.252), and time*-
hemisphere*group interactions (F =0.004, p =.947) were found.
3.2.2. Mean diffusivity
The analysis of MD revealed a signicant hemispheric effect (F =
4.988, p =.027) and time*group interaction (F =6.303, p =.013). No
signicant effects of time (F =2.253, p =.136), group (F =1.313, p =
.254) or time*hemisphere*group interaction (F =0.057, p =.812) were
found. Subsequent post-hoc testing showed signicantly higher MD in
the right UF (0.838E−3 ±0.004E−3, p =.022) compared to the left UF
(0.828E−3 ±0.003E−3) over both groups and timepoints. Further-
more, in the placebo group, right UF MD values were signicantly
increased at the post-intervention (0.845E−3 ±0.005E−3, p =.018)
compared to the baseline assessment (0.826E−3 ±0.004E−3), whereas
MD at post-intervention remained stable in the probiotics group (0.828
±0.004E−3, p =.904) compared to baseline (0.832E−3 ±0.005E−3)
(Fig. 1C).
3.3. Functional connectivity
The analysis including all seeds of interest showed a signicant
interaction in two different clusters located in the precuneus (x = +16 y
= − 52 z = +18, 89 voxels, p-FDR =0.0068, Fig. 2) and the left superior
parietal lobule extending to the left posterior supramarginal gyrus (x =
−44 y = − 42 z = +50, 61 voxels, p-FDR =0.0266). Post-hoc tests of the
precuneus cluster revealed signicant time*group interactions in the
subcallosal cortex, bilateral amygdala and hippocampus, and left tem-
poral pole (Fig. 2A–F). In these regions, the probiotics group showed
rsFC increases, whereas the placebo group showed rsFC decreases.
Baseline rsFC was only identical in both study groups in the left temporal
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Journal of Aective Disorders 324 (2023) 529–538
533
pole. Subsequent models correcting for baseline differences in all other
regions showed that the group effect was signicant in the right (F(1) =
4.24, p <.05) and left hippocampus (F(1) =6.1, p <.05), and left
amygdala (F(1) =10.31, p <.01), but not in the subcallosal cortex (F(1)
=1.63, p =.21) and right amygdala (F(1) =3.46, p =.07).
The cluster in the left superior parietal lobule (Fig. 3) had signicant
time*group interactions in rsFC originating from the subcallosal cortex,
left OFC, right amygdala and left hippocampus. Besides an increase of
rsFC in the left OFC (Fig. 3B), rsFC decreased in all other regions in the
probiotics group compared to placebo (Fig. 3A, C, D). Baseline rsFC was
not identical in the study groups in the subcallosal cortex rsFC but when
controlling for differences in baseline rsFC values, the group effect was
signicant (F(1) =8.09, p <.01).
3.4. Blood perfusion
The factorial ANOVA analysis showed no signicant group (F =
2.654, p =.109) and time (F =0.171, p =.681) effects, and no
group*time interaction (F =0.034, p =.854) in the subcallosal cortex.
There was also no signicant time (F =3.087, p =.081) and hemispheric
(F =1.051, p =.307) effect in the hippocampus, while the group effect
was signicant (F =5.415, p =.023). The placebo group demonstrated
signicantly higher mean CBF in the hippocampus (18.856 ±0.380 ml/
100 g/min, p =.003) compared to the probiotics group (17.099 ±
0.444 ml/100 g/min) across both time points. A signicant time effect
was found in the amygdala (F =7.198, p =.007); at the post-
intervention the mean CBF was signicantly higher (mean CBF =
14.642 ±0.421 ml/100 g/min, p =.007) compared to baseline (13.070
Fig. 2. Changes in resting-state functional connectivity (rsFC) over time for six connections with signicant interactions from (A) subcallosal cortex, (B) temporal
pole left, (C) hippocampus right, (D) hippocampus left, (E) amygdala right, (F) amygdala left to the cluster in the precuneus. The connection from subcallosal cortex
and right amygdala were not signicant after baseline correction. *Baseline differences between groups p <.05.
G. Yamanbaeva et al.

Journal of Aective Disorders 324 (2023) 529–538
534
±0.374 ml/100 g/min) (Supplementary Fig. 3). No other effects and
interactions were signicant in the amygdala, left and right insula, left
and right OFC, left and right temporal pole.
3.5. Relations to depressive symptoms
In the left UF within the probiotics group, changes in FA correlated
negatively with HAM-D changes (rho = − 0.662, p =.010) (Fig. 4A),
indicating greater HAM-D decreases with greater FA increases. On the
other hand, changes in MD in the left UF correlated positively with
HAM-D changes (rho =0.701, p =.005) (Fig. 4B), indicating greater
HAM-D decreases with greater MD decreases. Within the probiotics
group, FA changes correlated negatively with MD changes in the left UF
(rho = − 0.587, p =.027) (Fig. 4C) and in the right UF (rho = − 0.626, p
=.017) (Fig. 4D). No signicant correlations were found in the placebo
group.
Changes in rsFC from the right amygdala to the cluster in the supe-
rior parietal lobule correlated positively with HAM-D changes in the
probiotics group (rho =0.57, p =.032, Fig. 4E), indicating greater
symptom decreases with greater rsFC decreases. However, after cor-
recting for multiple testing, the correlation was not signicant anymore
(p-Bonferroni =0.12). Other correlations in the probiotics group and all
correlations in the placebo group were low and did not reach signi-
cance (Supplementary Table 1).
Changes of absolute CBF did not correlate with HAM-D changes in
any assessed region (Supplementary Table 2).
4. Discussion
A multi-modal neuroimaging approach was used to study the effects
of a probiotic add-on supplement on fronto-limbic network structure,
function, and blood perfusion in depressed patients. As rst major result,
we found an increased MD in the right UF within the placebo group with
no signicant change within the probiotics group, i,e, MD remained
stable. This stabilization of MD within the probiotic group positively
correlated with changes in depressive symptoms. Secondly, we found
changes over time between the study groups in rsFC between specic
fronto-limbic seed regions and a cluster in the precuneus and another in
the left superior parietal lobule. Decreases in right amygdala-superior
parietal lobule rsFC correlated positively with decreases in depressive
symptoms in the probiotics group.
Since lower FA and higher MD are attributed to a loss of micro-
structural integrity and neurodegeneration (Thomalla et al., 2004; van
Velzen et al., 2020), probiotics might prevent further neurodegeneration
given the fact that in the right UF the MD was signicantly increased in
the placebo group whereas in the probiotics group it remained stable, i.
e. the greater MD is the worse the neuronal integrity is preserved.
Notably, this effect was linked to the improvement of depressive
symptoms. In support, albeit not signicant, the left UF MD in the pro-
biotics group even showed a decrease, while it further increased in the
placebo group. FA has been attributed to the nerve ber arrangements,
axonal integrity, and the degree of axonal myelination (Basser and
Jones, 2002), whereas MD is considered to indicate atrophy and broad
cellular damages (Hutchinson et al., 2018; Immonen et al., 2009; Lai-
tinen et al., 2015; Pierpaoli et al., 1996; Putten et al., 2005). Interest-
ingly, decreased FA and increased MD are associated with neuronal
degeneration (Thomalla et al., 2004). Taken the abovementioned facts
together, our MD ndings suggest that the benecial clinical effect of
probiotic supplementation in depression (Schaub et al., 2022) may
partially be mediated by counteracting neuronal degeneration along the
Fig. 3. Changes in resting-state functional connectivity (rsFC) over time for four connections with signicant interactions from (A) subcallosal cortex, (B) orbito-
frontal cortex right, (C) hippocampus left (D) amygdala right to the cluster in the superior parietal lobule. *Baseline differences between groups p <.05.
G. Yamanbaeva et al.

Journal of Aective Disorders 324 (2023) 529–538
535
UF.
FA and MD are typically inversely correlated since myelination,
which facilitates directionality (i.e. increasing FA), also mediates a re-
striction to the overall movement (i.e. decreasing MD) (Green et al.,
2010; Niogi and Mukherjee, 2010). In our study, the signicant negative
correlation between FA and MD changes in both left and right UF within
the probiotics group indicates a subtle increase in the FA that is asso-
ciated with the reduction of depressive symptoms at least in the left UF
even though the time*group interaction in the FA was not signicant.
Taking into account that none of these correlations were found in the
placebo group, we propose that probiotics might not only prevent the
worsening of microstructural properties of the UF but also induce the
recovery of them revealed in the strong inverse correlation between FA
and MD and their association with the improvement of the depressive
symptoms.
Time*group interactions in rsFC from specied fronto-limbic regions
linked to the UF showed signicant clusters in the precuneus and the
superior parietal lobule, indicating that rsFC increased in connections to
the precuneus and decreased in connections to the superior parietal
lobule after probiotics intake. Decreased rsFC between the precuneus
and the subcallosal cortex has previously been shown in medication-
naive rst-episode adolescents with depression (Connolly et al., 2013).
Thus, the increase in this connection we found in the probiotics group
compared to placebo, might indicate a normalization of this connection
due to the probiotics. However, our result was not signicant after
controlling for baseline differences between the study groups.
Previously, increased rsFC has been found from the precuneus to the
hippocampus, as well as to different frontal regions and the middle
temporal gyrus in medication-naïve patients with rst-episode depres-
sion (Peng et al., 2015). This study also showed negative associations
between symptom severity and rsFC of the precuneus–temporo/parietal
junction as well as a positive association between symptom severity and
the precuneus–dorsomedial prefrontal cortex rsFC. In contrast to our
hypothesis, we could not nd any correlation between precuneus rsFC
and symptom changes over time. Generally, the precuneus is structur-
ally and functionally relevant in depression, showing volume loss
(Grieve et al., 2013) and altered rsFC as part of the default-mode
network (DMN) (Greicius et al., 2007; Kaiser et al., 2015; Li et al.,
2018). Previous results showed decreasing rsFC between lateral OFC and
precuneus in patients receiving medication while an increased rsFC has
been observed in unmedicated depressed patients (Cheng et al., 2018).
We could not nd a signicant time*group interaction effect in this OFC-
precuneus connection which might be explained with a general anti-
depressant effect of the medication that is independent from the pro-
biotics and was not of interest in this analysis.
In our second cluster located in the left superior parietal lobule, four
Fig. 4. Correlations of changes in depressive symp-
toms (HAM-D) and changes in neuroimaging mea-
surements. (A) Correlation between changes in
depressive symptoms and changes in fractional
anisotropy (FA) of the left uncinated fasciculus (UF)
in the probiotics group. (B) Correlation between
changes in depressive symptoms and changes in mean
diffusivity (MD) of the left UF in the probiotics group.
(C) Correlation between changes in FA and MD of the
left UF within the probiotics group. (D) Correlation
between changes in FA and MD of the right UF within
the probiotics group. (E) Correlation between changes
in depressive symptoms and changes in resting-state
functional connectivity (rsFC) from right amygdala
to the cluster in the left superior parietal lobule.
G. Yamanbaeva et al.

Journal of Aective Disorders 324 (2023) 529–538
536
of our seeds showed signicant interactions including the subcallosal
cortex, left OFC, right amygdala and left hippocampus. The superior
parietal lobule is involved in cognition including visually guided actions
in spatial processing (Sack, 2009) and it is also supposed to rearrange
information as part of the working memory (Koenigs et al., 2009).
However, the role of the superior parietal lobule in depression is not well
elucidated yet; its involvement has mostly been investigated as part of
the DMN that shows a hyperconnectivity in depression (Kaiser et al.,
2015). First-episode, drug-naive depressed patients exhibited a signi-
cantly lower magnetization transfer ratio (a quantitative measure of the
macromolecular structural integrity of brain tissue, to identify bio-
physical alterations) in the left superior parietal lobule compared to
healthy controls, which may be associated with the attentional and
cognitive dysfunction in depressed patients (Chen et al., 2016; Zhang
et al., 2018). The increasing rsFC from the superior parietal lobule to the
left OFC might be linked with cognitive functioning which has been
shown previously to be improved after probiotics treatment in depres-
sion (Kim et al., 2021). Furthermore, the OFC plays a special role in
decision making and emotions (Rolls et al., 2020; Wallis, 2007). In
contrast, the connectivity from subcortical regions (amygdala, hippo-
campus) and the subcallosal cortex to the cluster in the superior parietal
lobule decreases after the probiotics. The detected positive correlation in
the right amygdala-superior parietal lobule connectivity indicates that
greater rsFC decreases are associated with greater treatment response in
the probiotics but not in the placebo group. The decrease in this
connection of cognition-emotion relevant structures might be linked to
normal affect/reduced depressive symptoms. As altered rsFC from
amygdala to various brain regions has previously been shown in anti-
depressant treatments, it has been suggested that amygdala rsFC might
be a potential biomarker in depression (Connolly et al., 2017). Future
research could deepen the understanding of the probiotics’ effect on this
identied connection by additionally investigating working memory
functioning.
In depression, there is increased blood perfusion in the subgenual
ACC and lateral OFC, which normalizes in remission (Drevets, 2007).
Abnormalities in CBF of depressed patients were found in the dorsolat-
eral prefrontal cortex, rostral and ventral ACC, amygdala, and basal
ganglia (Bonne et al., 2003; Drevets, 2000; Mayberg et al., 1999): most
of the studies report reduced perfusion in these regions (Klemm et al.,
1996; Mayberg et al., 1994; Videbech, 2000), although contradicting
results have also been described reporting an increase in activity (Abou-
Saleh et al., 1999; Maes et al., 1993). In our study, we found a signicant
CBF increase over time in the amygdala in both groups, but the CBF
changes in the amygdala did not correlate with HAM-D changes in both
study groups. Taking into account the absent time*group interaction,
these ndings suggest that probiotics did not affect the blood perfusion
in the amygdala, or that the four-weeks probiotics intervention is not
sufcient long to see an effect. Moreover, the prevalent impact of the
antidepressant medication on the levels of the blood perfusion in the
assessed regions might be a reason for the absence of a reduced diffu-
sion. Nevertheless, it was reported that even a short-term antidepressant
therapy in depressed patients normalizes elevated CBF levels in the
amygdala (Godlewska et al., 2012; Smith and Cavanagh, 2005; Solleveld
et al., 2020) which is not consistent with the amygdala CBF elevation in
our patients who took antidepressant medication. However, there is a
certain contrariety in ndings related to the level of perfusion in limbic/
paralimbic regions including the amygdala (Drevets, 2002; Mayberg,
2003). More consistent single-photon emission computed tomography
in patients with MDD showed decreased perfusion (hypoperfusion) in
the dorsolateral prefrontal cortex, rostral and ventral anterior cingulate
cortex, basal ganglia, and amygdala in depressed patients (Smith and
Cavanagh, 2005). Also, the abnormal elevation in CBF (hyperperfusion)
in the amygdala has been associated with the severity of depression
(Drevets et al., 1992). Thus, the CBF elevation in our patients which was
not associated with changes in depressive symptoms nor with the pro-
biotic treatment must be interpreted carefully. In other words, to
determine whether after the antidepressants and probiotics the dynamic
of CBF level in the amygdala is positive (i.e. normalized) or negative, we
have to know whether initially there was a hyper- or hypoperfusion.
Further studies of this aspect with the larger study sample and
involvement of the healthy control would be required.
There are some limitations in this study that should be addressed in
future research. First, our sample size was moderate due to the multi-
modal complexity of the study design implying the participation of the
patients in two neuroimaging sessions and an intervention period of four
weeks, which produced a drop-out rate of >20 %. Also, some study
participants were excluded due to low compliance. Second, to see more
prominent changes, it could be advantageous to have a third MRI session
after eight weeks of intervention as the clinical effect was strongest at
the follow-up assessment (Schaub et al., 2022). The protective effect of
probiotics on the UF microstructural properties with their possible
consequent recovery could be more apparent then. Nevertheless, the
probiotic-induced prevention of further neurodegeneration along the UF
within the relatively short four-weeks intervention period and its rela-
tion to the improvement of depressive symptoms is intriguing.
This post-hoc analysis of an RCT explored the underlying neural
mechanisms of a probiotic add-on intervention in depressed patients.
The results suggest that the probiotics affect brain structure and function
in the fronto-limbic network and these effects are partly associated with
the decrease in depressive symptoms. Understanding the neural and
further biological mechanisms of probiotic supplementation along the
MGB axis on depressive symptoms in patients across the affective
spectrum may help to foster stratied treatment guidance by parsing
heterogeneity in the treatment responses. Large-scale studies may
further elucidate the precise mechanisms of probiotic supplementation
along the MGB axis including neural, immune, metabolic, and endocrine
pathways.
Conict of interest
The authors report no biomedical nancial interests or potential
conicts of interest.
Acknowledgments
The study was supported by the Gertrud Thalmann Foundation of the
University Psychiatric Clinics (UPK) Basel (SBo, UEL), the K¨
ampf-
B¨
otschi Foundation (UEL), the research fund junior researchers from
University of Basel (Appln 3MS1041, AS), the research fund of the UPK
Basel (AS) and the Stiftung zur F¨
orderung der gastroenterologischen und
allgemeinen klinischen Forschung sowie der medizinischen Bildaus-
wertung (AS). MENDES S.A., Switzerland, supplied the investigational
medicinal product. The funders had no role in the design and conduct of
the study; collection, management, analysis, and interpretation of the
data; preparation, review, or approval of the manuscript; and decision to
submit the manuscript for publication.
AS had full access to all the data in the study and takes responsibility
for the integrity of the data and the accuracy of the data analysis. LM,
CB, SBo, AS, and UEL designed the study. GY, ACS, ES, NS, CK, JPKD,
and AS contributed to the acquisition, analysis, or interpretation of data.
Statistical analyses were performed by GY and ACS. Administrative,
technical, or material support was provided by LM, SBr, SBo, AS, and
UEL. GY, ACS, and AS drafted the manuscript. All authors critically
reviewed the article and approved the nal manuscript.
T1-weighted structural MRI data of 50 patients at baseline were
recently published in the paper “Neural mapping of anhedonia across
psychiatric diagnoses: A transdiagnostic neuroimaging analysis”
(Schaub et al., 2021).
The authors report no biomedical nancial interests or potential
conicts of interest.
G. Yamanbaeva et al.

Journal of Aective Disorders 324 (2023) 529–538
537
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jad.2022.12.142.
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