Understanding gut feelings: Probiotics and cognition

In 2004, a seminal study demonstrated that germ-free mice show enhanced
reactivity to stress (Sudo et al., 2004). This study spurred research into what
is nowadays called the microbiota-gut-brain axis; i.e, the proposition that
microorganisms living in the gut may affect functioning of the central nervous
system (e.g. Foster & McVey Neufeld, 2013). Many animal studies have followed
confirming that gut-brain interactions have behavioural and mood effects
(Cryan & O*Mahony, 2011; Foster, Rinaman, & Cryan, 2017). For example, it was
shown that transplanting gut bacteria from a highly explorative mouse strain to
a more passive strain, and vice versa, altered behaviour in each mouse strain
showed that, together with the gut-microbiota, also behavioural traits had
swapped (Bercik et al., 2011). Other animal studies demonstrated, amongst
others, antidepressant properties of probiotic bacteria. Moreover, probiotics
administered to rats positively affected gut barrier integrity, cytokine
activity, and central HPA- and monoaminergic activity; biological processes
that have been implicated in depression (e.g. Gareau et al., 2007; Desbonnet et
al., 2008; Bravo et al., 2011; Gilbert et al., 2013, for a review see Desbonnet
et al., 2010).

Human studies have remained sparse, but a recent meta-analysis of 9 human
studies (Evrensel & Ceylan, 2015) provided support that manipulation of the
*microbiota-gut-brain axis* through the intake of probiotic food supplements
has anti-depressant effects, by reducing symptoms depression in healthy non-
depressed individuals. The fact that these effects are already observed in
non-depressed individuals is important, and dove-tails with recommendation of
the World Health Organization (2012) regarding the need for preventive
strategies for depression (Huang, Wang, & Hu, 2016).

A major limitation of the human literature on probiotic supplementation,
however, is that it has remained elusive by which mechanism probiotics yield
their beneficial effects. While a number of potential mechanisms have been
proposed on the basis of animal research, these mechanisms have received little
to no scrutiny in humans.

Translating these animal findings to humans is one of the aims of the current
study. The proposed project aims to replicate the aforementioned preliminary
findings (Evrensel & Ceylan, 2015; Steenbergen et al., 2015; de Noos et al.,
2015; Cryan & O*Mahony, 2011; Foster, Rinaman, & Cryan, 2017) and extend these
to include an investigation of the biological- and cognitive mechanisms by
which a probiotic intervention may have beneficial effects on mood.

Hence, the proposed study aims to:
1) Determine effects of probiotics on cognitive processes thought to signify
vulnerability to depression;
2) Test potentially mediating biological pathways herein.
Regarding aim 1, the study will focus on four cognitive domains.
a) cognitive reactivity/perseverance (CR; see above)
b) positive re-biasing in negative attentional processing; a mechanism
counteracting negative attentional bias.
c) Motivation (wanting) and reward (liking)-related learning (Felger &
Treadway, 2016)
d) affect recognition; recognition of affect-laden facial expressions has been
found to predict depression vulnerability (Pizzagalli, 2014; Bistricky, Ingram,
& Atchly, 2011; Boruke, Douglas, & Porter, 2010).
Regarding aim 2, the study will investigate four potential pathways identified
in animal studies (Cryan & Dinan, 2012; Desbonnet et al., 2008,2010), but which
received little scrutiny in humans:
a) gut permeability (GP), i.e., markers of leakage of bacterial products into
the circulation.
b) Through GP, and other routes, probiotics may affect inflammatory activity
(Mass, Kubera, & Leunis, 2008)
c) dysregulate activity of the hypothalamic-pituitary-adrenal (HPA) axis, which
both have been implicated in depression and cognitive functioning (Foster &
Neufeld, 2013; Pariante & Lightman, 2008).
d) Animal studies show that gut microbiota can increase amino acid metabolism
(Desbonnet et al., 2008, 2010), which may enhance brain neurotransmitter
availability (Silber & Schmidt, 2010).

In addition, we will account for factors known to affect or be affected by
these domains and pathways, specifically cognitive ability, fatigue, sleep
quality, dietary pattern, physical characteristics, microbiome profile, stool
characteristics, interaction between these factors, and changes herein (see
also Sections 8.1.2 and 8.1.3; Secondary and other parameters; Sandhu et al.,
2017; Budree et al., 2017).

The proposed study will follow a double-blind randomized, placebo-controlled,
between-subject design. A sample of 140 adults (18-75 years old) will be
included. All questionnaires and tasks, with the exception of a five-week mood
diary, a weekly food questionnaire, and the collection of a fecal sample, will
be completed during lab visits by the participants before and after the
intervention period of 35 days, during which participants will be asked to take
either 2 grams of Ecologic®Barrier (probiotics) or 2 grams of a neutral placebo
(standard carrier). For a detailed description of the study*s procedures, we
would like to refer to section 8.3. Participants (N=140, following Huang, Wang,
& Hu, 2016; see also section 4.3 for a detailed power analysis) will be
randomly assigned to either the probiotics (n=70) or the control (placebo)
group (n=70). Subjects will be treated according to the international
convention governing drug studies in human volunteers; i.e. the declaration of
Helsinki (1964) and its subsequent amendments.

Following Steenbergen and colleagues (2015) and de Noos and colleagues (2015),
the probiotic group will receive 2 grams freeze-dried powder of the probiotic
mixture Ecologic®Barrier daily for 35 days. (Winclove probiotics, the
Netherlands). Ecologic®Barrier (2.5 × 109 cfu/gram) contains the following
bacterial strains: Bifidobacterium bifidum W23, Bifidobacterium lactis W51 and
W52, Lactobacillus acidophilus W37, Lactobacillus brevis W63, Lactobacillus
casei W56, Lactobacillus salivarius W24, Lactococcus lactis W19 and Lactococcus
lactis W58. The placebo group will only receive the standard carrier used for
the probiotic product: i.e., 2 grams of maize starch for the same intervention
period. Participants in both groups will be asked to dissolve the carrier
products in lukewarm water before ingestion.

The burden of participation is modest and consists of one phone interview (to
screen for in- and exclusion criteria) and 2 visits to the laboratory, 35 days
apart. Each testing session will take no longer than 2.5 hours during which
questionnaires will be filled out and cognitive tests will be performed. At
both visits an 18 ml blood sample will be taken by a trained nurse or
phlebotomist, and supervised by a medical doctor. Standard measurements will
include heart rate and blood pressure (using an automated monitor) and
assessment of anthropomorphic characteristics (i.e., weight, height,
skin-folds, waist- and hip circumference). Participants will collect
(self-produced) fecal matter in a specially designed collection system that
requires very little effort from the participants. Between visits, participants
will be asked to keep a diary and to take the required food supplement. The
dosage of Ecologic*Barrier (the brand name of the probiotic supplement
utilized) is deemed safe ; all strains hold qualified presumption of safety
(QPS) status and are approved by the European Food Safety Authority (EFSA).
This food supplement is freely accessible to consumers on the market and the
negligible health risks in the general population have been well-established.