Correspondence
Psychiatry
Adult Hippocampal Neurogenesis Is Regulated by
the Microbiome
To the Editor:
Accumulating evidence suggests that the microbiota plays a
key role in the brain and behavior (1–4). Alterations in the gut
microbiota-brain axis have been implicated in stress-related
disorders such as depression and anxiety, in neurodevelop-
mental disorders such as autism, and in cognitive functioning
(1,4–6). Neurogenesis, the birth of new neurons, in the adult
hippocampus is thought to play an important role in learning
and memory and responses to stress and antidepressant
drugs (7–9). Germ-free (GF) mice have grown up without
having ever been exposed to microorganisms and are an
excellent tool to probe the role of the microbiota in health and
disease. Several studies have suggested that some brain
processes that are regulated by adult hippocampal neurogen-
esis are also affected in GF mice (6,10), and altered concen-
trations of substrates known to influence adult hippocampal
neurogenesis, such as corticosterone, serotonin, brain-derived
neurotrophic factor, and proinflammatory cytokines, have
been reported in these mice (2,11). However, whether adult
hippocampal neurogenesis is altered in GF mice has not yet
been reported.
We assessed adult hippocampal cytogenesis and neuro-
genesis in GF mice and control mice (conventionally colonized
[CC]) and investigated whether such alterations in adulthood
would be prevented by microbial colonization of GF mice
during early life (germ-free–colonized [GF-C] mice). To assess
cell proliferation, 10-week-old mice were injected with bromo-
deoxyuridine (BrdU) to label proliferating cells (150 mg/kg, i.p.)
and euthanized 2 hours later. To assess the survival of newly
born cells and newly born neurons, 7-week-old mice received
four injections of BrdU (75 mg/kg, i.p., every 2 hours) as
previously described (12,13) and were euthanized at 10 weeks
of age. GF Swiss Webster mice were bred and maintained in a
GF facility at University College Cork, whereas control CC
Swiss Webster mice were bred and housed in the conven-
tional animal facility (11). At weaning, a group of GF mice was
colonized with microbiota (GF-C mice) by removing the mice
from the GF facility at weaning (3 weeks) and placing them in
the conventional animal facility in cages with bedding and
fecal matter from CC mice as previously described (11). To
measure the proliferation and survival of newly born cells in the
subgranular zone (SGZ) across the entire rostrocaudal axis of
the hippocampus, BrdU immunohistochemistry was con-
ducted as previously described (12,13). The number of surviv-
ing newly born neurons (neurogenesis) in the SGZ was
determined by counting the number of BrdU-positive/neuronal
nuclei (NeuN)-positive cells in every sixth section (35-μm
coronal free-floating sections) (1:250 rat anti-BrdU ab6326;
Abcam, Cambridge, United Kingdom; mouse anti-neuronal
nucleus (NeuN) 1:100, MAB377; Merck Millipore, Merck KGaA,
Darmstadt, Germany). The total number of cells was multiplied
by 6 to get an estimate for the entire SGZ. All data were
analyzed by one-way analysis of variance followed by Fisher’s
SEE COMMENTARY ON PAGE
http://dx.doi.org/10.1016/j.biopsych.2014.12.023
ISSN: 0006-3223
Biological
least significant difference post hoc test for group-wise
comparisons.
Across the total SGZ, cell proliferation (Figure 1A) was
increased in GF and GF-C mice, although the effect did not
reach statistical significance [F2,18 5 3.248, p 5 .062] (n 5 7).
The survival of newly born cells (Figure 1B) was significantly
increased in GF mice [F2,15 5 7.862, p 5 .005] (GF vs. CC, p 5
.009; n 5 6), and this effect was not prevented by postweaning
microbial colonization (GF-C vs. CC, p 5 .002). The survival of
newly born neurons (Figure 1C,D) was significantly increased
in GF mice [F2,15 5 4.291, p 5 .034] (GF vs. CC, p 5 .045;
n 5 6), and this effect was not prevented by postweaning
microbial colonization (GF-C vs. CC, p 5 .014).
Accumulating evidence suggests that the hippocampus is
functionally segregated along its longitudinal axis into dorsal
and ventral regions whereby the dorsal hippocampus plays a
preferential role in spatial learning and memory, whereas the
ventral hippocampus preferentially regulates anxiety and the
stress response (14). More recent reports indicate that neuro-
genesis might be differentially regulated in these subregions
(12-15). We found that increased survival of newly born cells
and newly born neurons occurred preferentially in the dorsal
hippocampus (anterior–posterior [AP] 2.94 to AP 22.30
according to Paxinos and Franklin’s atlas of the mouse brain
(16) rather than the ventral hippocampus (AP 22.46 to AP
23.80 according to Paxinos and Franklin’s atlas of the mouse
brain (16): dorsal hippocampus (newly born cells [F2,15 5
8.431, p 5 .004]; newly born neurons [F2,15 5 4.574,
p 5 .028]); ventral hippocampus (newly born cells [F2,15 5
1.943, p 5 .178]; newly born neurons [F2,15 5 .141, p 5 .870]).
In summary, GF mice exhibit increased adult hippocampal
neurogenesis, and this effect occurs predominantly in the
dorsal hippocampus. Postweaning microbial colonization of
GF mice did not prevent changes in adult hippocampal
neurogenesis, suggesting that there is a critical window in
early life during which microbial colonization influences adult
hippocampal neurogenesis. Although the mechanisms under-
lying these effects are unclear, these data further support the
emerging hypothesis that the microbiota influences brain
function.
Ebere S. Ogbonnaya
Gerard Clarke
Fergus Shanahan
Timothy G. Dinan
John F. Cryan
Olivia F. O’Leary
Acknowledgments and Disclosures
This work was supported by Science Foundation Ireland Grant Nos. SFI/12/
RC/2273, 02/CE/B124, and 07/CE/B1368 (Alimentary Pharmabiotic Centre);
Health Research Awards Grant Nos. HRA_POR/2011/23 (TGD) and HRA-
POR-2014-647 (GC, TGD); Brain and Behavior Research Foundation
National Alliance for Research on Schizophrenia and Depression Young
Investigator Grant No. 20771 (GC); European FP7 (KBBE.2013.2.2-02;
MyNewGut: Microbiome influence on energy balance and brain develop-
ment function put into action to tackle diet-related diseases and behaviour)
(TGD, JFC); Health Research Board of Ireland Grant No. HRA_POR/2012/32
& 2015 Society of Biological Psychiatry e7
Biological Psychiatry August 15, 2015; 78:e7–e9 www.sobp.org/journal
Biological
Psychiatry Correspondence

Figure 1. Germ-free mice exhibit increased adult hippocampal neurogenesis. Germ-free and germ-free–colonized mice exhibit a trend for increased cell
proliferation as measured by bromodeoxyuridine immunohistochemistry (A). The survival of newly born cells is significantly increased in the dorsal, but not
ventral, hippocampus of germ-free and germ-free–colonized mice (B). The survival of newly born neurons is increased in germ-free and germ-free–colonized
mice (C), and this effect occurs preferentially in the dorsal hippocampus (C, D—upper panels) and not the ventral hippocampus (C, D—lower panels). *p ,
.05, **p , .01 significantly different from conventionally colonized control mice. BrdU, bromodeoxyuridine; CC, conventionally colonized; DHI, dorsal
hippocampus; GF, germ-free; GF-C, germ-free colonized; NeuN, neuronal nucleus; VHI, ventral hippocampus.

(JFC TGD); Science Foundation Ireland Investigator Award Grant No. 12/IA/
1537 (JFC); and European Community’s Seventh Framework Programme
Grant No. FP7/2007-2013 under Grant Agreement No. 278948 (Transla-
tional Adolescent and Childhood Therapeutic Interventions in Compulsive
Syndrome) (JFC).
References
We thank Dr. Daniela Felice, Ms. Marie O’Donovan, Mr. Patrick
Fitzgerald, Frances O’Brien, and Dr. Michael Lyons for technical assistance.
ESO reports no biomedical financial interests or potential conflicts of
interest. FS, TGD, and JFC are principal investigators in the Alimentary
Pharmabiotic Centre, University College Cork. GC and OFO are faculty
members of the Alimentary Pharmabiotic Centre. The Alimentary Pharma-
biotic Centre has conducted research funded by Pfizer, GlaxoSmithKline,
Proctor & Gamble, Mead Johnson, Suntory Wellness, and Cremo. OFO has
been supported by the University College Cork strategic research fund and
has been an invited speaker at a meeting organized by Janssen. TGD has
been an invited speaker at meetings organized by Servier, Lundbeck,
Janssen, and AstraZeneca. JFC has been an invited speaker at meetings
organized by Mead Johnson and Yakult.
Article Information
From the Departments of Anatomy and Neuroscience (ESO, JFC, OFO),
Psychiatry (GC, TGD), and Medicine (FS) and Alimentary Pharmabiotic
Centre (GC, FS, TGD, JFC, OFO), University College Cork, Cork, Ireland.
Authors JFC and OFO contributed equally to this work.
Address correspondence to John F. Cryan, Ph.D; E-mail: j.cryan@ucc.ie.
Received Dec 22, 2014; accepted Dec 30, 2014.
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