The Journal of Neuroscience, September 21, 2011 • 31(38):13469 –13484 • 13469

Behavioral/Systems/Cognitive

Stimulation of Entorhinal Cortex Promotes Adult
Neurogenesis and Facilitates Spatial Memory
Scellig S. D. Stone,1,2,3,5 Cátia M. Teixeira,1,6 Loren M. DeVito,1 Kirill Zaslavsky,1 Sheena A. Josselyn,1,2,4
Andres M. Lozano,2,3,5 and Paul W. Frankland1,2,4
1Program in Neurosciences and Mental Health, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8, 2Institute of Medical Science, 3Divisionof Neurosurgery, and 4Department of Physiology, University of Toronto, Toronto, Ontario, Canada, M5S 1A8, 5Division of Neurosurgery, Toronto Western
Hospital, Toronto, Ontario, Canada, M5T 2S8, and 6Graduate Program in Areas of Basic and Applied Biology, University of Porto, 4050-465 Porto, Portugal

Deep brain stimulation (DBS) is an established therapeutic modality for the treatment of movement disorders and an emerging therapeutic approach for the treatment of disorders of mood and thought. For example, recently we have shown that DBS of the fornix may
ameliorate cognitive decline associated with dementia. However, like other applications of DBS, the mechanisms mediating these clinicaleffects are unknown. As DBS modulates neurophysiological activity in targeted brain regions, DBS might influence cognitive function via
activity-dependent regulation of hippocampal neurogenesis. Using stimulation parameters analogous to clinical high-frequency DBS,
here we addressed this question in mice. We found that acute stimulation of the entorhinal cortex (EC) transiently promoted proliferationin the dentate gyrus (DG). Cells generated as a consequence of stimulation differentiated into neurons, survived for at least several weeks,
and acquired normal dentate granule cell (DGC) morphology. Importantly, stimulation-induced promotion of neurogenesis was limited
to the DG and not associated with changes in apoptotic cell death. Using immunohistochemical approaches, we found that, once sufficiently mature, these stimulation-induced neurons integrated into hippocampal circuits supporting water-maze memory. Finally, formation of water-maze memory was facilitated 6 weeks (but not 1 week) after bilateral stimulation of the EC. The delay-dependent natureof these effects matches the maturation-dependent integration of adult-generated DGCs into dentate circuits supporting water-mazememory. Furthermore, because the beneficial effects of EC stimulation were prevented by blocking neurogenesis, this suggests a causal
relationship between stimulation-induced promotion of adult neurogenesis and enhanced spatial memory.

Introduction
Deep brain stimulation (DBS) uses surgically implanted electrodes to deliver electrical stimulation to precisely targeted areasin the brain. To date, ⬎60,000 patients have been implanted withdeep brain electrodes (Ponce and Lozano, 2010), and its predominant application has been in the treatment of movement disorders, most commonly Parkinson’s disease (Koller et al., 1997;
Vidailhet et al., 2005; Deuschl et al., 2006). However, studies havealso begun to explore its potential application to a widening arrayof neurologic and psychiatric conditions, including disorders ofReceived June 18, 2011; revised July 25, 2011; accepted July 29, 2011.
Author contributions: S.S.D.S., S.A.J., A.M.L., and P.W.F. designed research; S.S.D.S., C.M.T., L.M.D., and K.Z.
performed research; S.S.D.S., L.M.D., and S.A.J. analyzed data; S.S.D.S., A.M.L., and P.W.F. wrote the paper.
The authors declare no conflicts of interest.
This work was supported by Canadian Institutes of Health Research Grants MOP-86762 and IAP-102234 (P.W.F.).
S.S.D.S. was supported by a Canadian Institutes of Health Research Fellowship, the Department of Surgery’s Surgeon
Scientist Program at the University of Toronto, and a Michael J. Fox Foundation Fellowship. C.M.T. received support
from the Graduate Program in Areas of Basic and Applied Biology and the Portuguese Foundation for Science andTechnology. S.A.J. holds a Tier 2 Canada Research Chair in Molecular and Cellular Cognition. A.M.L. holds a Tier 1
Canada Research Chair in Neuroscience and the R.R. Tasker Chair in Functional Neurosurgery at the University ofToronto. P.W.F. holds a Tier 2 Canada Research Chair in Cognitive Neurobiology. We thank Berge Minassian forproviding tracer compound and Masanori Sakaguchi and Maithe Arruda-Carvalho for technical assistance.
Correspondence should be addressed to Paul W. Frankland, Neurosciences and Mental Health, The Hospital for
Sick Children, 555 University Avenue, 6018 McMaster Building, Toronto, ON, Canada, M5G 1X8. E-mail:paul.frankland@sickkids.ca.
DOI:10.1523/JNEUROSCI.3100-11.2011
Copyright © 2011 the authors 0270-6474/11/3113469-16$15.00/0

mood and thought. For example, DBS of limbic circuits has
shown promise in treating refractory depression (Mayberg et al.,2005), whereas DBS of basal ganglia circuitry has been used in thetreatment of obsessive compulsive disorder (Mallet et al., 2008)
and Tourette syndrome (Shahed et al., 2007). Moreover, we recently reported a phase 1 trial investigating DBS of the limbic
system for the treatment of dementia/cognitive impairment(Laxton et al., 2010). In this trial, DBS of the fornix was associatedwith arrest or slowing of cognitive decline in certain patients,
measured by two different scales of cognitive functioning. Although the mechanism(s) of action underlying these clinicaleffects, and DBS in general, remain(s) poorly understood (Kringelbach et al., 2007), it is clear that DBS modulates the activity oftargeted brain circuits (Davis et al., 1997; Mayberg et al., 2005).
For instance, in our trial, fornix stimulation strongly activated the
hippocampus and parahippocampal gyrus, including the entorhinal cortex (EC) (Laxton et al., 2010).
One potential mechanism through which DBS might influence cognitive function is via activity-dependent regulation ofhippocampal neurogenesis. New neurons are continuously added tothe adult mammalian dentate gyrus (DG), a subregion of the hippocampus that plays an essential role in memory formation (Mingand Song, 2005; Zhao et al., 2008). These adult-generated dentate
granule cells (DGCs) eventually develop similar cellular phenotypes
to developmentally generated DGCs (Laplagne et al., 2006, 2007),
and, once sufficiently mature, are thought to contribute to the for-

13470 • J. Neurosci., September 21, 2011 • 31(38):13469 –13484

mation of hippocampus-dependent memory (Shors, 2008; Deng etal., 2010). Consistent with the idea that activity regulates neuron
production in the adult DG, stimulation of limbic targets in rodents[e.g., the anterior thalamic nucleus (Toda et al., 2008; Encinas et al.,2011), EC/perforant path (Bruel-Jungerman et al., 2006; Chun et al.,2006; Kitamura et al., 2010), or mossy fibers (Derrick et al., 2000)]
promotes the proliferation and/or survival of adult-generated DGCs
in vivo. Although these studies demonstrate that stimulation of hippocampal afferents can reliably increase the production of new neurons, whether these stimulation-induced increases in neurogenesis
impact hippocampal function is not known. Our experiments indicate that new granule cells, born as a consequence of EC stimulation,
mature normally and assume functional roles in hippocampal
circuits supporting spatial memory. Furthermore, stimulationinduced promotion of hippocampal neurogenesis facilitates spatial
memory formation, suggesting that enhanced neurogenesis is one
mechanism via which DBS may have pro-cognitive effects.

Materials and Methods
Mice. Male offspring from a cross between C57BL/6NTacfBr and
129Svev mice (Taconic) were used in these experiments. All mice werebred in the colony at The Hospital for Sick Children and housed ingroups of three to five mice per cage. They were maintained on a 12 h
light/dark cycle with access to food and water ad libitum and were 8 weeks
old at the start of experiments. Behavioral procedures were conductedduring the light phase of the cycle, blind to the treatment condition of themouse and according to protocols approved by the Animal Care Committee at The Hospital for Sick Children.
Stereotactic surgery. Mice were pretreated with atropine sulfate (0.1mg/kg, i.p.), anesthetized with chloral hydrate (400 mg/kg, i.p.), andplaced in a stereotactic frame. The scalp was incised and skull hole(s)drilled. Targets with coordinates relative to bregma in the anteroposterior, mediolateral, and dorsoventral planes were as follows (in mm): (1)EC bordering on the lateral and medial entorhinal areas, specifically near thejunction of the ventral intermediate, medial, and caudal entorhinal fields(van Groen, 2001) [⫺4.0, 3.0, 5.1]; (2) DG [⫺2.0, 1.6, 2.0]; (3) fourth andfifth cerebellar lobules [⫺6.2, 1.0, 2.0] (Paxinos and Franklin, 2000).
Fluorescent anterograde tracer labeling. An anterograde tracer (fluorescent-labeled 10 kDa dextran [fluoro-ruby D-1817; Invitrogen]; 0.82
mg/ml) was injected unilaterally into the EC via a glass micropipette(outer diameter, ⬃50 ␮m) connected to a Hamilton syringe. Injection
volume (1.5 ␮l) and rate (0.15 ␮l/min) were controlled by an infusion
pump (SP100i; WPI). The micropipette was left in place for an additional
10 min to ensure complete diffusion of tracer.
Brain electrical stimulation. Electrical stimulation was delivered via
concentric bipolar electrodes (CBASC75; FHC), the detailed electricalproperties of which have been described previously (Gimsa et al., 2005,
2006). Electrode integrity was verified by impedance measurement.
Stacked contacts include a 100-␮m-long ⫻ 125-␮m-diameter stainlesssteel outer pole, 100 ␮m separation, and 100-␮m-long ⫻ 25-␮mdiameter platinum/iridium inner pole. Targeting accuracy was refined to⫾0.5 mm in the anteroposterior and mediolateral planes and ⫾0.25 mm
in the dorsoventral plane during pilot experiments. Stimulation was applied with a clinical screener (model 3628; Medtronic) using frequency(130 Hz) and pulse width (90 ␮s, square wave) settings approximating
high-frequency DBS used in clinical practice (Volkmann et al., 2006).
Current (0 –500 ␮A), duration (30 –120 min), and laterality (unilateralor bilateral) of stimulation varied by experiment. Charge density, calculated using electrode geometry (McCreery et al., 1990; Gimsa et al., 2005,
2006), was kept below 30 ␮C/cm 2/phase to minimize the risk ofstimulation-induced neural injury (McCreery et al., 1990).
Retroviral labeling of new neurons. New neurons were labeled by CAG
promoter-driven green fluorescent protein (GFP) expression after infection with a replication-deficient retroviral vector (based on the Moloneymurine leukemia virus) (Tashiro et al., 2006). Viral vector was preparedby transfecting Platinum-gp cells with two plasmids containing an amphotropic envelope (vesicular stomatitis virus-glycoprotein) and theStone et al. • Entorhinal Cortex Stimulation Enhances Neurogenesis and Memorytransgene (pCAG–GFP), followed by collection through ultraspeed centrifugation. Platinum-E cells were then infected to generate a stable virusproducing cell line, and concentrated virus solution was obtained byultraspeed centrifugation (average of 3.5 ⫻ 10 9 IU/ml). Viral vectors
were injected bilaterally into the DG via a glass micropipette (outer diameter, ⬃50 ␮m) connected to a Hamilton syringe. Injection volume(1.5 ␮l/side) and rate (0.12 ␮l/min) were controlled by an infusion pump
(SP100i; WPI). Micropipettes were left in place for an additional 10 min
to ensure complete diffusion of virus.
BrdU, CldU, and IdU administration. The thymidine analogs 5-bromo-2⬘deoxyuridine (BrdU; Sigma), 5-iodo-2⬘-deoxyuridine (IdU; MP Biomedicals), or 5-chloro-2⬘-deoxyuridine (CldU; Sigma) were dissolved in 0.1 M
PBS and heated to 50 – 60°C, at a concentration of 10 mg/ml. Intraperitoneal
BrdU injection dosages and dosing intervals are described below (see Experimental procedures). IdU and CldU were given intraperitoneally at equimolar concentrations to 50 mg/kg BrdU (corresponding to 57.5 mg/kg IdU and42.5 mg/kg CldU) three times per day (8 h apart) for 3 consecutive days as
indicated below (see Experimental procedures).
Temozolomide administration. Temozolomide (TMZ) (Sigma) was
dissolved in dimethylsulfoxide (DMSO), diluted in PBS to a concentration of 2.5 mg/ml (10% DMSO), and administered intraperitoneally at adose of 25 mg/kg once daily for 3 consecutive days according to a previous protocol (Garthe et al., 2009). Vehicle solution (V) was the identical
DMSO/PBS solution but without TMZ and was administered in volumes
consistent with TMZ dosing.
Water-maze apparatus and procedures. The apparatus and behavioral
procedures have been described previously (Teixeira et al., 2006). In
brief, a circular water-maze tank (120 cm diameter, 50 cm deep), locatedin a dimly lit room, was filled to a depth of 40 cm with water (maintained
at 28 ⫾ 1°C and made opaque by adding white, nontoxic paint). A
circular escape platform (10 cm diameter) was submerged 0.5 cm below
the water surface, in a fixed position in one quadrant. White curtains with
distinct cues painted on them surrounded the pool, each ⱖ1 m from thepool perimeter.
Before training, mice were individually handled for 2 min each day
over 7 consecutive days. Mice were trained with one of two protocols:
standard training over 5 d with six training trials per d (presented in two
blocks of three trials; interblock interval, ⬃ 1 h; intertrial interval, ⬃15 s)
or undertraining over 3 d with three trials per day (intertrial interval,⬃15 s). On each trial, mice were placed into the pool, facing the wall, in
one of four start locations (the order of which was pseudorandomly
varied throughout training). The trial was complete once the mousefound the platform or 60 s had elapsed. If the mouse failed to find theplatform on a given trial, the experimenter guided the mouse onto theplatform. After the completion of training, spatial memory was assessedin a series of three probe tests with an intertest interval of ⬃3 min. In thistest, the platform was removed from the pool, and the mouse was allowed
60 s to search for it.
Behavioral data from training trials and probe tests were acquired andanalyzed using an automated tracking system (Actimetrics). Generaltraining measures included latency to reach the platform and path
length. Search strategies during training were analyzed using numericalparameters from swim tracking data (adapted from Garthe et al., 2009),
and the respective predominant search strategy for each trial was objectively classified by a criterion-based algorithm. Trials were classified as
one of the following ordered strategies (tracking criteria in parentheses):(1) “direct swim,” characterized by a maintained heading toward theplatform (⬍15 cm path length or heading ⬍22.5° away from platform ateach 5 cm point starting at 15 cm path length); (2) “focal search,” characterized by highly localized search near the platform (⬎50% trial in 15
cm radius target zone, centered on the platform location); (3) “directed
search,” characterized by a preference for a corridor toward the platform
or platform quadrant (⬍50% trial outside a 50-cm-wide corridor fromstart point to platform and ⬎20% in 25 cm target zone, or ⬎40% intarget quadrant); (4) “chaining,” characterized by searching near thecorrect radial distance of the platform to the wall (⬎75% trial 20 –50 cm
from pool center, ⬍15% within 10 cm of wall, and ⬍10% within 20 cm ofpool center); (5) “scanning,” characterized by a preference for the central
pool area in which distal cue visibility is maximal (⬎50% trial within 35

Stone et al. • Entorhinal Cortex Stimulation Enhances Neurogenesis and Memory

cm of pool center); (6) “thigmotaxis,” characterized by maintaining close
proximity to the wall (⬎70% trial within 10 cm of wall); (7) “perseverance,” characterized by an erroneous preference for a non-target area(⬎60% trial in one or ⬎75% in two adjacent non-target quadrant(s) and⬎750 cm path length); and (8) “random search,” characterized by noother discernable strategy (remaining unclassified trials).
Probe test performance was quantified in three ways: (1) the amountof time mice searched the target zone versus the average of the three otherequivalent zones in other areas of the pool [zone radius was either 15 or
20 cm (for the TMZ experiment)], (2) the number of platform location
crossings versus the average of the three other equivalent platform locations in other areas of the pool, and (3) the frequency with which mice
visited areas of the pool represented as a density plot (or heat map, withhot colors corresponding to more frequent visits) generated using Matlab (MathWorks).
Immunohistochemistry. Mice were perfused transcardially with 0.1 M
PBS and 4% paraformaldehyde (PFA). Brains were removed, fixed overnight in PFA, and transferred to 0.1 M PBS. Fifty micrometer horizontal(for some tracer analyses) or coronal sections were cut using a vibratome(VT1200S; Leica). TUNEL staining was performed using the DeadEnd
Calorimetric TUNEL System (Promega) and cresyl violet counterstaining. For other analyses, the following primary antibodies were used: rabbit polyclonal anti-Fos (1:1000; Calbiochem), rat monoclonal anti-BrdU
for BrdU and CldU specifically (1:500; Accurate Chemicals), mousemonoclonal anti-BrdU for IdU specifically (1:1000; BD Biosciences),mouse monoclonal anti-NeuN (1:1000; Millipore Bioscience Research
Reagents), rabbit polyclonal anti-GFP (1:500; Invitrogen), and Alexa
Fluor 488-conjugated mouse monoclonal anti-NeuN (1:1000; Millipore Bioscience Research Reagents). Detailed BrdU/IdU/CldU/Fos/
NeuN staining procedures have been described previously (Stone etal., 2010). In brief, sections were washed and incubated for 48 h at 4°C
with primary antibodies and then for 2 h at 20°C with the following
fluorescent-conjugated or biotinylated secondary antibodies: Alexa
Fluor-488 anti-rat (1:500; Invitrogen), Alexa Fluor-568 anti-mouse(1:500; Invitrogen), Alexa Fluor-568 anti-rabbit (1:500; Invitrogen),
Rhodamine Red-X-conjugated anti-mouse (1:500; Jackson ImmunoResearch), FITC-conjugated anti-rat (1:500; Jackson ImmunoResearch), or
Biotin-SP-conjugated anti-rabbit (1:500; Jackson ImmunoResearch). Biotinylated antibody signals were visualized using Alexa Fluor-conjugatedstreptavidin (Invitrogen). Sections were slide mounted with Permafluor
anti-fade medium (Lipshaw Immunon).
Imaging and quantification. All images were acquired using epifluorescent (BX61; Olympus) or confocal laser-scanning (LSM710; Carl Zeiss)microscopes and displayed as maximum-intensity projections of Z-stack
images created using Image-Pro (Media Cybernetics) or ZEN (Carl
Zeiss) software. A laser pinhole setting of 1 Airy unit and Z-stack image
spacing of 0.5–1.0 ␮m (spanning the regions of interest) were used for all
confocal imaging. These produced serial optical slices of 0.8 ␮m and 1.0
␮m under 100⫻ and 40⫻ objectives, respectively.
Fluorescent tracer distribution was qualitatively assessed from survey
images acquired using 10 – 40⫻ objectives on the epifluorescent microscope. The presence of tracer within neuronal projections was verified
using confocal microscopy. DG regional analysis was performed using
serial sections covering the entire anteroposterior extent of the DG,
grouped by thirds as anterior, middle, or posterior.
BrdU ⫹, CldU ⫹, IdU ⫹, GFP ⫹, Fos ⫹, and CldU⫹ or IdU ⫹ (XdU⫹)/
Fos ⫹ cells were quantified using a 40⫻ objective on the epifluorescent
microscope. DG total cell numbers were estimated stereologically perside using systematic random sampling fractions of 1⁄4 serially cut coronal
sections covering the entire anteroposterior extent of the DG. The tops of
nuclei served as unique characteristic points for exhaustive direct cellcounting using Stereo Investigator (MicroBrightField) software. Sample
totals were then multiplied by the inverse sampling rate (4) to give total
DG estimates. After identification under the epifluorescent microscope,
apparent double-labeled cells were confirmed by confocal microscopy.
Area densities of subventricular zone (SVZ) BrdU ⫹ cells were derived
using a method modified from Bath et al. (2008). A systematic random
sample fraction of 1⁄4 serially cut coronal sections, covering ⬃1.54 – 0.50
mm relative to bregma, was used. All BrdU ⫹ nuclei within 0.1 mm of the

J. Neurosci., September 21, 2011 • 31(38):13469 –13484 • 13471

lateral ventricle ependymal surfaces were counted per side, marking thetops of nuclei as unique characteristic counting points, using a 40⫻
objective on the epifluorescent microscope and Stereo Investigator software. For each section counted, reference lines tracing each ependymal
surface were drawn using Stereo Investigator software. Area densities
were then represented by dividing total cell counts by total reference line
lengths for each side [giving BrdU ⫹ cells/(1.0 mm reference line ⫻ 0.1
mm subventricular zone depth), or BrdU ⫹ cells/0.1 mm 2].
To estimate the proportions of IdU ⫹ and CldU ⫹ cells that were colabeled with NeuN after unilateral surgery, DG regions of interest wererandomly selected from five mice and analyzed using confocal microscopy. A total of 300 IdU ⫹ and 300 CldU ⫹ cells were analyzed (i.e., 30
cells per side per mouse). The proportions of BrdU ⫹ cells colabeled with
NeuN after bilateral surgery and drug treatment were identically derived
from five to eight mice per group.
Neuron tracings and spine counts were manually collected from 0.5
␮m spaced Z-stack images acquired with a 100⫻ objective on the epifluorescent microscope and using Neurolucida (MicroBrightField) software. In each of three mice in the 6 week group, six ipsilateral and six
contralateral neurons were analyzed (for a total of 18 ipsilateral and 18
contralateral neurons from three mice). Dendritic architecture andspines were quantified for each neuron in its entirety (i.e., spines werecounted along the entire dendrite length and on all dendrite branches foreach neuron). Dendritic architecture was classified as aberrant if there
was a dendritic process with spines pointing toward the hilus, and granule cells were considered ectopic if located more than two cell layers awayfrom the inner border of the granule cell layer (Jessberger et al., 2007).
Total dendrite length, nodes, and linear spine density (total spines divided by total dendrite length for each branch order) were quantified
using Neurolucida software. Axon distribution was qualitatively assessedfrom survey images of the CA3 region using confocal microscopy.
Experimental procedures. To characterize EC projections to the DG,
mice received unilateral injections of fluorescent tracer into the EC (n ⫽5) and were perfused 7 d later (Table 1).
Stimulation-induced DG activation was assessed by placing mice (n ⫽6) under anesthesia for 3 h before surgery. Unilateral EC stimulation wasdelivered for 1 h (50 ␮A). Anesthesia was maintained for an additionalhour before perfusion.
Stimulation-induced effects on DG proliferation and apoptosis were
determined after unilateral EC stimulation (50 ␮A for 1 h). After stimulation, mice were treated with a single 200 mg/kg BrdU injection 1 (n ⫽5), 3 (n ⫽ 4), 5 (n ⫽ 4), or 7 (n ⫽ 4) d later and perfused 24 h after
injection. Additional nonstimulated mice underwent electrode insertion,with no current delivery, followed by BrdU 1 (n ⫽ 5), 3 (n ⫽ 6), 5 (n ⫽ 4),
or 7 (n ⫽ 4) d postoperatively.
To examine the effects of varied stimulation durations and currents oncell proliferation, mice received unilateral EC stimulation. Three days
later, they received a single 200 mg/kg BrdU injection and were perfused
24 h later. To examine the impact of stimulation duration on proliferation, 50 ␮A was delivered for 30 (n ⫽ 5), 60 (n ⫽ 4), or 120 (n ⫽ 5) min.
To examine the effect of current intensity on proliferation, mice received
50 (n ⫽ 4), 250 (n ⫽ 5), or 500 (n ⫽ 5) ␮A stimulation for 1 h.
The proportion of stimulation-induced newly born cells adopting aneuronal phenotype was assessed after unilateral EC stimulation (50 ␮A
for 1 h, n ⫽ 5). Mice received IdU injections during the period of
stimulation-induced increased proliferation (postoperative days 3–5),
CldU injections during a similar period of baseline proliferation (postoperative days 7–9), and were perfused ⬃10 weeks later.
To assess stimulation-induced effects on cell survival, mice weretreated twice daily with 100 mg/kg BrdU injections (8 h apart) for 3
consecutive days, 1, 10, or 30 d (n ⫽ 8 each) before unilateral EC stimulation (50 ␮A for 1 h), and perfused 3 weeks postoperatively.
Subventricular zone proliferation after unilateral EC stimulation and
DG proliferation after unilateral cerebellar stimulation were assessed inseparate experiments using single 200 mg/kg BrdU injections 3 d after
stimulation (50 ␮A for 1 h). Both experiments included stimulated andnonstimulated groups (n ⫽ 4 each) perfused 24 h after BrdU injection.
Retroviral-mediated labeling of adult-born DGCs was performed bybilateral viral solution injections into the DG 3 d after unilateral EC

Stone et al. • Entorhinal Cortex Stimulation Enhances Neurogenesis and Memory

13472 • J. Neurosci., September 21, 2011 • 31(38):13469 –13484

Table 1. Experimental procedures
Experiment (Figure)

Treatment

Anterograde tracer (1a– d)Unilateral EC tracer
infusion
Unilateral EC S
Unilateral EC S, NS

S-induced Fos (1e– h)
S-induced DG proliferation (2),apoptosis (4f– h)
S-induced DG survival (3d,e)S-induced SVZ proliferation (4a– c)
Cerebellar S (4d,e)Retroviral labeling of DGCs (5)S-induced DG neurogenesis (3a– c)
and DGC integration (6)S effects on spatial memory (7, 8)

Unilateral EC S, NS
Unilateral EC S, NS
Unilateral cerebellar
S, NS
Unilateral EC S, NS
Unilateral EC S, NS
Bilateral EC S, NS

TMZ/S effects on spatial memory (9) TMZ or V before
bilateral EC S, NS

Cell labeling

Perfusion

Maze training

Maze probe

7 d after infusion

BrdU 1, 3, 5, or 7 d after S

1 h after S
24 h after BrdU

BrdU 1, 10, or 30 d before S
BrdU 3 d after S
BrdU 3 d after S

3 weeks after S
24 h after BrdU
24 h after BrdU

Bilateral DG retrovirus infu- 1 or 6 weeks after infusion
sion 3 d after S
IdU 3–5 d, CldU 7–9 d after S ⬃10 weeks after XdU
Standard training ⬃1 or 6
⬃10 weeks after S
weeks after XdU
Undertraining 1.5 or 6.5 weeks 1 h after training or 1.5 weeks after S
after, or 1 d before S
BrdU 3–5 d or ⬃7 weeks⬃7 weeks after S
Undertraining 6.5 weeks after S 1 h after training
after SNS, Nonstimulation; S, stimulation.

stimulation (50 ␮A for 1 h). Mice were perfused 1 or 6 weeks (n ⫽ 3 each)later.
The long-term survival of stimulation-induced newly born neurons
and their integration rate into spatial memory networks, relative to similarly aged cells, was assessed after unilateral EC stimulation (50 ␮A for
1 h). Mice received IdU injections during the period of stimulationinduced increased proliferation (postoperative days 3–5) and CldU injections during a similar period of baseline proliferation (postoperativedays 7–9). Water-maze standard training occurred 6 weeks (n ⫽ 17,
includes 5 mice used for NeuN phenotype experiment) or 1 week (n ⫽15) later, followed by probe testing 4 or 9 weeks later, respectively. Mice
were perfused 90 min after probe testing.
EC stimulation-induced changes in spatial memory performance wereassessed in bilaterally stimulated (50 ␮A for 1 h, n ⫽ 25) or nonstimulated (n ⫽ 26) mice who underwent water maze training (undertraining
protocol) 6.5 weeks later and subsequent probe testing 1 h after the lasttraining trial. In a second experiment, stimulated (n ⫽ 25) and nonstimulated (n ⫽ 26) mice were trained (undertraining protocol) 1.5
weeks after surgery. In the third experiment, mice were first trained(undertraining protocol), followed 1 d later by stimulation (n ⫽ 24) or
nonstimulation (n ⫽ 24) surgery and subsequent probe testing 1 week
later. In the final experiment, animals received V or TMZ injections
followed on the next day by bilateral stimulation (V-S, n ⫽ 24; TMZ-S,n ⫽ 24) or nonstimulation (V-NS, n ⫽ 17; TMZ-NS, n ⫽ 17). Six and ahalf weeks after surgery, animals were trained in the water maze (undertraining protocol) and received a probe test 1 h after the last training trial.
To assess the proportion of stimulation-induced newly born cells adopting a neuronal phenotype, BrdU was given intraperitoneally at a dose of
50 mg/kg per injection three times per day (8 h apart) for 3 consecutive
days to subsets of animals in each group: on postoperative days 3–5 (V-S,n ⫽ 8; TMZ-S, n ⫽ 5; V-NS, n ⫽ 8; TMZ-NS, n ⫽ 5) during the period of
stimulation-induced increased proliferation or for 3 d after the completion of water-maze testing (V-S, n ⫽ 5; TMZ-S, n ⫽ 7; V-NS, n ⫽ 7;
TMZ-NS, n ⫽ 5) ⬃7 weeks postoperatively.
Statistical analyses. Behavioral (excluding swim strategy) and cellcount (excluding scatter plots) data are presented as mean ⫾ SEM andwere evaluated using ANOVAs or t tests (two-tailed). Duncan’s multiple
range test was used for post hoc analyses of significant main effects orinteractions when appropriate. Swim strategy frequencies were compared across trials within groups using Friedman’s test and over all trials
between groups using Pearson’s ␹ 2 test for independence. Scatter plot
data were analyzed using Pearson’s correlation.

Results
Entorhinal cortex projects ipsilaterally to the DG
The EC provides the main afferent input to the DG (Amaral andLavenex, 2007). Because neurogenesis is regulated by neural ac-

tivity (Zhao et al., 2008), stimulation of these afferents provides a
direct method to increase adult neurogenesis. To characterize
this projection, we first injected a fluorescent, anterograde tracer
into the EC (Fig. 1a). Our injection site was centered on thejunction of the ventral intermediate, medial, and caudal entorhinal fields (van Groen, 2001) (Fig. 1b). Viewed in horizontal sections, labeled axons extended anteromedially from the injection
site. They coursed through the angular bundle region to thesubiculum, past CA1–CA3, and around the hippocampal fissure
via the stratum lacunosum moleculare, eventually reaching themolecular layer of the DG. Focal dilations and club-shaped terminals were evident along this path and were especially dense in
the outer two-thirds of the molecular layer, a pattern that is consistent with terminal labeling of fibers from both the medial andlateral perforant paths (Amaral and Lavenex, 2007). Fibers also
coursed along the alveus, consistent with direct alvear projections
to the subiculum and CA1 (Amaral and Lavenex, 2007). In addition, projections also reached the presubiculum and, to a lesser
extent, the parasubiculum (van Groen and Wyss, 1990). Labelingwas virtually restricted to the side ipsilateral to the injection site,
consistent with previous reports that the EC projection to the DG
is predominantly ipsilateral (van Groen et al., 2002, 2003). Furthermore, terminal labeling was densest in the middle and posterior regions of the DG (Fig. 1c,d), consistent with known
topographical organization of EC–DG connections (van Groenet al., 2002, 2003).
We next asked whether stimulation of the EC would increase
activity in the DG. To do this, we lowered an electrode into thesame EC site and delivered high-frequency stimulation for 1 h
under chloral hydrate anesthesia. Our stimulation parameters(50 ␮A, 90 ␮s pulse width delivered at 130 Hz) were chosen tomodel those used for high-frequency DBS clinically (Volkmann
et al., 2006). Sixty minutes after the completion of stimulation,
we assessed expression of the activity-regulated gene c-Fos (Fig.
1e). Consistent with the tracing experiment, Fos expression waselevated in the DG granule cell layer, suggesting that stimulation
led to sustained activation of DGCs. The increase in Fos expression was limited to the side ipsilateral to the site of stimulation(Fig. 1f,g), consistent with the finding that the EC–DG projection
is predominantly unilateral. Moreover, increased Fos expression
was essentially confined to the middle and posterior portions of
the DG, a pattern that matches the topographical organization ofStone et al. • Entorhinal Cortex Stimulation Enhances Neurogenesis and Memory

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critically, there was a significant Side ⫻Region interaction (F(2,10) ⫽ 6.13, p ⬍0.05), reflecting more pronounced changes in
Fos expression in the ipsilateral middle
and posterior regions of the DG (Duncan’s post hoc tests comparing ipsilateraland contralateral sides, p values ⬍0.05).
EC stimulation transiently increases
adult neurogenesis in the DG
We next asked whether stimulation of theEC would increase proliferative activity in
the DG. To do this, mice were injectedwith the proliferation marker BrdU, 1, 3,
5, or 7 d after stimulation of the EC.
Twenty-four hours later, BrdU incorporation was quantified in the DG (Fig. 2a).
EC stimulation increased the number of
BrdU-labeled cells (Fig. 2b,c). An ANOVA,
with Side (ipsilateral, contralateral) as awithin-subjects variable and Stimulation(stimulated, nonstimulated) and Day (1,3, 5, 7) as between-subject variables,
revealed that these increases were anatomically specific and delay dependent (significant three-way Side ⫻ Stimulation ⫻ Day
interaction, F(3,28) ⫽ 3.90, p ⬍ 0.05): increased BrdU incorporation was limited tothe ipsilateral side and to groups injectedwith BrdU 3–5 d after stimulation (Duncan’s post hoc tests, p values ⬍0.05). These
results indicate that the pro-proliferative effects of EC stimulation are not immediate
but rather emerge after a few days. In addition, contralateral to the electrode site, BrdU
incorporation was equivalent in both stimulated and nonstimulated mice (Duncan’s
post hoc tests, p values ⬎0.05). This indicates that stimulation does not modulate
proliferation contralateral to the electrodesite, and therefore the contralateral side
may be used as a within-animal control
for basal rates of proliferation after unilateral stimulation of the EC. Finally, to exFigure 1. Entorhinal cortex projects ipsilaterally to the DG. a, Tracer distribution was examined 7 d after unilateral EC target plore the topographical specificity of theinjection (n ⫽ 5). b, NeuN-stained (green) horizontal section (top 2 panels; scale bar, 200 ␮m) showing tracer (red) distribution stimulation-induced changes in proliferaipsilateral (I, left panels) and contralateral (C, right panels) to EC target (dotted oval) injection (al, alveus; ca1–3, cornu ammonis tion in detail, we divided the DG into equal
regions 1–3; hf, hippocampal fissure; lec, lateral EC; mec, medial EC; ml, molecular layer; pa, parasubiculum; pr, presubiculum; s, thirds from the anterior-to-posterior pole.
subiculum; slm, stratum lacunosum moleculare). Squares in top panels indicate perforant path regions magnified in corresponding In the 3 d group, we found that increased
bottom panels (scale bar, 5 ␮m). c, Sagittal schematic indicating anterior (Ant), middle, and posterior (Post) thirds of DG. d, proliferation was confined to the middle
Representative DG coronal sections ipsilateral (I, left panels) and contralateral (C, right panels) to tracer injection. Molecular layer and posterior ipsilateral DG (Fig. 2d; Side ⫻afferents were labeled in the middle and posterior (bottom 2 panels) regions of the ipsilateral DG (gcl, granule cell layer; numbers Region ANOVA, significant interaction,
indicate anteroposterior distance to bregma; scale bar, 50 ␮m). e, Fos expression was examined 1 h after unilateral stimulation
F(2,10) ⫽ 7.73, p ⬍ 0.01; Duncan’s post hoc(n ⫽ 6). f, DAPI (blue)-stained coronal section showing DG Fos (red) expression ipsilateral (I, left panel) and contralateral (C, righttests
comparing ipsilateral and contralateral
panel) to stimulation (scale bar, 50 ␮m). g, Fos expression increased in middle (Mid) and posterior (Post) regions of the ipsilateral(I), but not contralateral (C), DG. h, Representative coronal sections ipsilateral (I, left panels) and contralateral (C, right panels) to sides, p values ⬍0.05). This topographicalstimulation from anterior (top panels), middle (middle panels), and posterior (bottom panels) DG regions (as per schematic in c; organization is consistent with the tracing
and immediate early gene analyses.
scale bar, 20 ␮m). *p ⬍ 0.05.
We next evaluated how stimulation
duration and intensity impacts DG prolifterminal labeling after injection of anterograde tracer into theeration. First, to examine the impact of duration, mice were stimsame site (Fig. 1g,h). An ANOVA with Side (ipsilateral, contralatulated for 30, 60, or 120 min using the same stimulationeral) and Region (anterior, middle, posterior) as within-subject
parameters as before. They were then injected with BrdU 3 d later,
factors supported the anatomical specificity of these effects. Most
and, as previously, BrdU incorporation was assessed 24 h later.

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Stone et al. • Entorhinal Cortex Stimulation Enhances Neurogenesis and Memory

Increasing stimulation duration increased
proliferation ipsilateral to the electrodesite (Fig. 2e; Side ⫻ Duration ANOVA,
significant interaction, F(2,11) ⫽ 5.39, p ⬍0.05): there were more BrdU-labeled cells
after 60 min (compared with 30 min)stimulation (Duncan’s post hoc test, comparing 30 and 60 min on ipsilateral side;p ⬍ 0.01) but no additional benefit of further increasing stimulation duration to
120 min (Duncan’s post hoc test, comparing 60 and 120 min on ipsilateral side; p ⫽0.42). Second, to examine the impact of
current intensity on proliferation, micereceived 50, 250, or 500 ␮A of stimulation for 60 min. They were then injectedwith BrdU 3 d later, and BrdU incorporation was assessed 24 h later. Althoughstimulation-induced changes in granulecell layer/subgranular zone proliferation
were limited to the ipsilateral side, there
were no effects of stimulation intensity(Fig. 2f; Side ⫻ Intensity ANOVA, significant main effect of Side only, F(1,11) ⫽45.76, p ⬍ 0.01), suggesting that there
might be a ceiling on these stimulationinduced changes in proliferation. Indeed,
at 250 and 500 ␮A, BrdU-labeled cells
were additionally found in the hilus andmolecular layer, suggesting that highintensity stimulation may additionallypromote proliferation of non-neuronal Figure 2. EC stimulation increases proliferation in adult DG. a, Separate groups of mice were injected with BrdU at different
cell types (Madsen et al., 2005) and/or ab- delays after unilateral stimulation or electrode insertion only (n ⫽ 4 – 6 per group). b, BrdU ⫹ cells (green) ipsilateral (I, left
errant neurogenesis analogous to that panels) and contralateral (C, right panels) to electrode site (gcl, granule cell layer; h, hilus; DAPI counterstain; scale bars: top, 50
seen after seizures (Parent et al., 1997, ␮m; bottom, 10 ␮m). c, BrdU incorporation in the ipsilateral (I) and contralateral (C) DG in stimulated (S) versus or nonstimulated2006).
(NS) mice at different postsurgery delays. d, Stimulation increased proliferation in the middle (Mid) and posterior (Post) ipsilateral
Under basal conditions, ⬃70 –90% of DG regions. e, Proliferation was increased after 60 or 120 min of stimulation (BrdU was injected 3 d after stimulation) (n ⫽ 4 –5 percells generated in the adult subgranular group). f, Proliferation was equivalently increased in the ipsilateral DG after stimulation at 50, 250, and 500 ␮A (BrdU was injectedzone differentiate into neurons (Brandt et 3 d after stimulation) (n ⫽ 4 –5 per group). *p ⬍ 0.05, **p ⬍ 0.01.
al., 2003). To evaluate whether a similar
proportion of cells differentiate into neuthat observed after a short survival period (1 d; Fig. 2c), suggestrons after stimulation of the EC, mice were treated with equimoing that cells produced as a consequence of stimulation survive
lar doses of IdU and then CldU, two chemically related thymidine
for at least several weeks.
analogs. IdU and CldU are recognized by different antibodies andWe found that stimulation of the EC transiently increased thetherefore may be used to label separate cohorts of cells in the same
future production of neurons in the DG. To evaluate whether the
animal (Vega and Peterson, 2005; Stone et al., 2010; Tronel et al.,same stimulation promotes survival of existing, adult-generated2010). In this experiment, IdU injections occurred 3–5 d after
cells, we next treated mice with the proliferation marker BrdU
stimulation, a poststimulation time point corresponding to theand then stimulated 1, 10, or 30 d later (Fig. 3d). The numbers ofpeak in stimulation-induced changes in proliferation, and CldU
BrdU-labeled cells were increased ipsilaterally (vs contralaterally)
injections occurred 7–9 d after stimulation, a poststimulationin the 10 d (planned comparison, paired t test, t(8) ⫽ 9.31, p ⬍time point when proliferation rates have returned to baseline0.01) group but not in the 1 d (planned comparison, paired t test,(Fig. 3a). Ten weeks later, there were no differences in the prot(8) ⫽ 0.43, p ⫽ 0.68) or 30 d (planned comparison, paired t test,
portion of IdU- and CldU-labeled cells expressing the neuronalt(8) ⫽ 0.38, p ⫽ 0.72) groups (Fig. 3e; one-way ANOVA, signifimarker NeuN, either ipsilateral or contralateral to the electrodecant effect of Group, F(2,21) ⫽ 5.46, p ⬍ 0.05; Duncan’s post hocsite (Fig. 3b,c; Side ⫻ Thymidine Analog ANOVA, main effects
tests, p values ⬍0.05). Consistent with previous reports (Brueland interaction p values ⬎0.05), indicating that stimulation did
Jungerman et al., 2006; Kitamura et al., 2010), these data indicate
not alter the fate of newly generated cells. Importantly, the numthat EC stimulation has a modest pro-survival effect on existing
ber of IdU-labeled cells was increased on the side ipsilateral to theadult-generated cells in the hippocampus. That these effects were
stimulation site (298.40 ⫾ 14.94) compared with IdU-labeled
specific to the 10 d group is consistent with the idea that 1-tocells in the contralateral DG (186.72 ⫾ 11.74) and CldU-labeled
3-week-old neurons are especially sensitive to factors that either
cells in the ipsilateral (192.04 ⫾ 12.29) and contralateralpromote (e.g., environmental enrichment) or reduce (e.g., stress)(186.53 ⫾ 11.55) DG. The magnitude of the relative increase aftersurvival (Zhao et al., 2008).
this extended survival period (⬃10 weeks) is comparable with

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J. Neurosci., September 21, 2011 • 31(38):13469 –13484 • 13475

Ma et al., 2009). Using our lower-current
intensity, we next evaluated whether localized stimulation elsewhere in the brain
might increase adult neurogenesis in the
DG. To do this, we targeted the fourth andfifth cerebellar lobules, regions that do notsend direct afferent input to the DG (Fig.
4d). BrdU incorporation 3 d after stimulation was unaltered by cerebellum stimulation (compared with a nonstimulated
control group) (Fig. 4e; Side ⫻ Stimulation ANOVA, main effects and interaction p values ⬎0.05), suggesting that
low-intensity stimulation of non-afferent
sites does not regulate adult neurogenesis
in the DG.
Third, it is possible that stimulation of
the EC might induce cell death in the DG,
leading to a compensatory increase in
adult neurogenesis (Gould and Tanapat,1997). Therefore, to evaluate the possibility that changes in neurogenesis are secondary to altered levels of DGC death, we
additionally stained tissue for TUNEL, amarker of programmed cell death, instimulated or control mice 2, 4, 6, or 8 dafter stimulation. Although there were regional differences in TUNEL counts, withlevels highest in the granule cell layer andlowest in the hilus, no such differenceswere observed between the ipsilateral
Figure 3. Survival and differentiation of stimulation-induced neurons. a, After unilateral stimulation (n ⫽ 5), mice were and contralateral sides, stimulated andinjected with IdU (during the period of increased proliferation) and CldU (once proliferation returned to baseline). b, Representa- nonstimulated groups, or days after stimtive confocal image of IdU ⫹ and CldU ⫹ DG cells colabeled with NeuN (scale bar, 20 ␮m), ipsilateral to stimulation. c, Similar ulation (Fig. 4f– h; Side ⫻ Region ⫻ Stimproportions of IdU ⫹ and CldU ⫹ cells were NeuN ⫹ ipsilateral (I) and contralateral (C) to electrode site. d, Separate groups of mice ulation ⫻ Delay ANOVA, significant
were injected with BrdU at different delays before stimulation (n ⫽ 8 per group). e, There were more BrdU ⫹ cells ipsilateral to the main effect of Region only, F(2,28) ⫽stimulation site in the mice treated with BrdU 10 d before surgery. **p ⬍ 0.01.
193.94, p ⬍ 0.01). Together, these datasuggest that our low-intensity EC stimuSpecificity of stimulation-induced increase in proliferation
lation does not induce apoptotic cell death in the DG and thereIn the above experiments, we identified a set of conditions
fore exclude the possibility that changes in levels of adult
whereby EC stimulation produces an approximate 1.5-fold toneurogenesis are secondary to stimulation-induced cell death.
2-fold increase in adult neurogenesis. We next explored the specificity of the effects of stimulation.
After stimulation, new neurons mature normallyFirst, the subgranular zone of the hippocampus is one of two
We found that EC stimulation increases adult neurogenesis in the
major neurogenic regions in the adult brain. Does stimulation of
DG. Because these newly generated neurons survive several
the EC additionally regulate neurogenesis in the subventricular
weeks, this raises the possibility that they become functionallyzone? To address this, additional groups of mice were treatedintegrated into hippocampal circuits. To evaluate this possibility,with BrdU 3 d after stimulation of the EC (Fig. 4a). Twenty-four
we used a retroviral vector strategy to label dividing neural proh later, BrdU-labeled cells were evident in the subependymal regenitors and their progeny with GFP (Tashiro et al., 2006). Begion of the subventricular zone (Fig. 4b). There were similarcause GFP is expressed throughout the cell body and processes,
numbers of BrdU-labeled cells in both the stimulation and nonusing this strategy, it is possible to track morphological changes
stimulation groups, and no additional differences between thein newborn cells at different stages after stimulation. Accordipsilateral and contralateral sides (Fig. 4c; Side ⫻ Stimulation
ingly, mice received unilateral stimulation and then, 3 d later,
ANOVA, main effects and interaction p values ⬎0.05), suggestbilateral infusion of retrovirus encoding GFP into the DG. Thising that EC stimulation specifically regulates hippocampal and3 d poststimulation time point corresponds to the peak of
not olfactory adult neurogenesis. Similarly, the pro-neurogenic
stimulation-induced changes in proliferation. Mice were killed
effects after stimulation of another limbic target, the anterior
either 1 or 6 weeks later.
thalamic nucleus, are limited to the DG (Encinas et al., 2011).
In the 1 week group (Fig. 5a), there were approximately twice
This contrasts with the biregional neurogenic effect seen after
as many GFP-labeled cells per section on the ipsilateral (27.09 ⫾generalized neural stimulation by seizures (Parent et al., 2002).
5.20) versus contralateral (13.95 ⫾ 2.38) side to the stimulation
Second, high-intensity, generalized electrical activation of thesite (Fig. 5b; paired t test, t(2) ⫽ 4.64, p ⬍ 0.05), reflecting
stimulation-induced changes in proliferation. Both the magnibrain (e.g., electroconvulsive shock, seizures) also increases adulttude and the anatomical specificity of this increase replicate ourneurogenesis in the DG (Madsen et al., 2000; Parent et al., 2002;

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Stone et al. • Entorhinal Cortex Stimulation Enhances Neurogenesis and Memory

BrdU incorporation data. Most of GFPlabeled cells were located in the subgranular zone or innermost part of the granulecell layer and had short, aspiny, processes
running either parallel to or, occasionally,
perpendicular to the granule cell layer. In
rare instances, these perpendicular processes extended through the granule cell
layer (but never into the molecular layer).
No axon fibers were visible in CA3. Consistent with previous reports (Zhao et al.,2006; Jessberger et al., 2007), the morphology of these GFP-labeled cells resembled immature neurons. A small minorityof cells (⬃2%) were ectopically located in
the hilus or deep within the granule cell
layer. Importantly, the frequency of theseectopically located cells was similar ipsilateral (1.77%) and contralateral (1.74%)
to the electrode site (paired t test, t(2) ⫽0.05, p ⫽ 0.96), indicating that stimulation did not alter the normal migrationand early stages of integration of adultgenerated neurons in the DG.
Six weeks after infection (Fig. 5c), there
were many GFP-labeled cells resembling
mature neurons. As before, there were approximately twice as many GFP-labeledcells on the ipsilateral (4.47 ⫾ 0.19) versus
contralateral (2.11 ⫾ 0.17) side to thestimulation site (Fig. 5d; paired t test,
t(2) ⫽ 16.28, p ⬍ 0.01), suggesting thata, Mice were injected with BrdU 3 d after unilateralcells, produced as a consequence of stim- Figure 4. Specificity of EC stimulation effects on hippocampal neurogenesis.
stimulation or electrode insertion only (n ⫽ 4 per group). b, BrdU ⫹ (green) cells in the subventricular zone (DAPI counterstain)
ulation, survive for at least several weeks. ipsilateral (I, left panel) and contralateral (C, right panel) to unilateral EC stimulation (scale bar, 50 ␮m). c, There were no
The vast majority of GFP-labeled cells differences between area density counts of BrdU ⫹ cells in stimulated (S) or nonstimulated (NS) mice either in the ipsilateral (I) or
were located in the subgranular zone or contralateral (C) SVZ. d, In a separate experiment, mice were injected with BrdU 3 d after unilateral cerebellar stimulation or
innermost part of the granule cell layer. electrode insertion only (n ⫽ 4 per group). e, There were no differences between the total BrdU ⫹ cell counts in stimulated (S)Typically, dendritic processes extended or nonstimulated (NS) mice in either the ipsilateral (I) or contralateral (C) DG. f– h, TUNEL counts were similar in the ipsilateral andthrough the granule cell layer with mini- contralateral granule cell layer (f ), molecular layer (g), or hilus (h) in stimulated (S) versus nonstimulated (NS) groups.
mal or no branching but arborized extentantly, the frequency was similar on both the ipsilateral (1.79%)
sively in the molecular layer. Furthermore, there were abundantand contralateral (2.08%) sides (paired t test, t(2) ⫽ 0.19, p ⫽spines on these apical dendritic processes, especially within the0.87), suggesting that stimulation does not promote the aberrant
molecular layer, suggesting that these newly generated neurons
integration of adult-generated neurons into the DG.
had become synaptically integrated into DG circuitry. GFPlabeled axonal fibers were also present in the CA3 region, consisNew neurons functionally integrate into hippocampal
tent with previous reports suggesting that, by 6 weeks of age,memory circuits
adult-generated neurons have established efferent connections
The morphological analyses indicate that new neurons, generwith CA3 (Zhao et al., 2006; Toni et al., 2008). Most importantly,
ated as a consequence of stimulation, integrate into hippocampal
apical dendrite branch morphology was similar on both the ipsicircuitry. Using a water-maze task, we next asked whether, once
lateral and contralateral sides to the electrode site, with equivaintegrated, these adult-generated neurons play a functional rolelent total dendrite length (Fig. 5e; paired t test, t(2) ⫽ 0.50, p ⫽0.67) and nodes per neuron (Fig. 5f; paired t test, t(2) ⫽ 0.09,
in hippocampus-mediated spatial learning. To address this, micep ⫽ 0.94). Furthermore, spine density on apical dendritic
were treated with equimolar doses of IdU and then CldU at difbranches was similar both ipsilateral and contralateral to the elecferent times after unilateral stimulation of the EC. As before, IdU
trode site (Fig. 5g; Side ⫻ Branch Order ANOVA, significant
injections occurred 3–5 d after stimulation (a time point corremain effect of Branch Order only, F(4,2) ⫽ 34.24, p ⬍ 0.01).
sponding to the peak in stimulation-induced changes in proliferTogether, these analyses suggest that stimulation promotes neuation), and CldU injections occurred 7–9 d after stimulation (arogenesis and that neurons produced as a consequence of stimutime point when proliferation rates have returned to baseline).
lation acquire normal morphology and likely establish normal
Six weeks later, mice were trained in the water maze, and thenafferent and efferent connections. Similar to the 1 week postinspatial memory was assessed in a probe test 4 weeks later (Fig. 6a).
fection group, a small minority of GFP-labeled cells were ectopBoth acquisition and expression of water-maze memories engage
ically located. The low incidence is consistent with previous
the DG, and engagement of dentate granule neurons after memreports (Parent et al., 1997; Jessberger et al., 2007), and, imporory recall may be assessed by quantifying expression of activity-

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J. Neurosci., September 21, 2011 • 31(38):13469 –13484 • 13477

Figure 5. Morphology of adult-generated granule cells. a, Unilaterally stimulated mice received bilateral GFP-retrovirus injections into the DG 3 d after stimulation (during the period of increased
proliferation) and were examined 1 week later (n ⫽ 3). b, Representative images ipsilateral (I, left 3 panels) and contralateral (C, right 3 panels) to stimulation at low (top panels; scale bar, 50 ␮m)
and high (bottom panels; scale bar, 10 ␮m) magnification showing immature-appearing GFP ⫹ (green) neurons (red, NeuN counterstain). c, A separate group was examined 6 weeks after retroviral
injections (n ⫽ 3). d, Representative images ipsilateral (I, left 6 panels) and contralateral (C, right 6 panels) to stimulation. Low (top row; scale bar, 50 ␮m) and high (middle row; scale bar, 20 ␮m)
magnification images show examples of mature-appearing neurons. GFP ⫹ axons and terminals were present bilaterally in CA3 (bottom row left panels; scale bar, 20 ␮m). High-magnification
images (bottom row right panels; scale bar, 10 ␮m) show relatively aspiny proximal (P, top panels), and spiny distal (D, bottom panels) portions of apical dendrites. e– g, Similar total dendrite
length (e), average nodes per neuron (f ), and progressive increase in linear spine density by branch order (g) ipsilateral versus contralateral to stimulation.

regulated genes such as c-Fos (Kee et al., 2007a). Therefore, should
stimulation increase the pool of functional new neurons, then the
proportion of adult-generated neurons included in the populationof activated (Fos-labeled) neurons should increase. Such an increase
should be specific to neurons generated 3–5 d after stimulation (i.e.,
IdU-labeled cells) and, because stimulation is unilateral, occur only
on the side ipsilateral to the electrode site.
In the probe test, mice searched selectively, spending moretime in the region of the pool that formerly contained the platform (Fig. 6b; paired t test, t(16) ⫽ 5.53, p ⬍ 0.01). Consistent with
our previous experiments, stimulation of the EC led to an increase in adult neurogenesis. As expected, this increase wasspecific to the ipsilateral side and corresponded to new cells generated 3–5 d (i.e., IdU-labeled) after stimulation (Fig. 6c,d; Side ⫻Thymidine Analog ANOVA, significant interaction, F(1,16) ⫽167.76, p ⬍ 0.01; Duncan’s post hoc tests, p ⬍ 0.01). After theprobe test, Fos expression was equivalent both ipsilateral andcontralateral to the stimulation site, suggesting equivalent levelsof activation (Fig. 6e; paired t test, t(16) ⫽ 0.98, p ⫽ 0.34). Within
this population of Fos-labeled neurons, there were many IdU- or
CldU-labeled cells. Most importantly, stimulation increased the

proportion of adult-generated neurons included in this population of activated (Fos-labeled) neurons. Critically, this increase
was specific to neurons generated 3–5 d after stimulation (i.e.,
IdU-labeled cells) and occurred only on the side ipsilateral to theelectrode site (Fig. 6f; Side ⫻ Thymidine Analog ANOVA, significant interaction, F(1,16) ⫽ 11.33, p ⬍ 0.01). Furthermore, thisincrease was proportional to the magnitude of the stimulationinduced increase in adult neurogenesis (Fig. 6g; r ⫽ 0.58, p ⬍ 0.05).
Therefore, these data suggest that EC stimulation increases the production of new neurons and that these new neurons assume functional roles in hippocampal circuits mediating memory.
The integration of adult-generated neurons into hippocampal
memory circuits is maturation dependent, with new neurons notcontributing in maximal numbers until they are ⬎5 weeks of age(Kee et al., 2007b; Stone et al., 2010). Therefore, stimulationinduced increases in the contribution of adult-generated neurons
should not occur if there is only a brief delay between stimulationand training. Accordingly, we repeated the above experiment but
trained mice 1 week (rather than 6 weeks) after IdU/CldU injections (Fig. 6h). In the probe test 9 weeks later, mice searchedselectively in the region of the pool formerly containing the plat-

13478 • J. Neurosci., September 21, 2011 • 31(38):13469 –13484

form (Fig. 6i; paired t test, t(14) ⫽ 8.16, p ⬍0.01). As before, stimulation of the EC led
to an increase in adult neurogenesis. This
increase was specific to the ipsilateral side
and corresponded to new neurons generated 3–5 d (i.e., IdU-labeled) after stimulation (Fig. 6j; Side ⫻ Thymidine Analog
ANOVA, significant interaction, F(1,14) ⫽33.48, p ⬍ 0.01; Duncan’s post hoc tests,p ⬍ 0.01). After the probe tests, Fos expression was equivalent both ipsilateraland contralateral to the stimulation site,
suggesting equivalent levels of activation(Fig. 6k; paired t test, t(14) ⫽ 0.14, p ⫽0.89). However, this Fos-labeled population of dentate granule neurons contained
few IdU- or CldU-labeled cells (Fig. 6l;
Side ⫻ Thymidine Analog ANOVA, nosignificant interaction, F(1,14) ⫽ 0.02, p ⫽0.89). Furthermore, there was no relationship between stimulation-induced increases
in neurogenesis and the proportion of
adult-generated neurons included in thispopulation of activated (Fos-labeled) neurons (Fig. 6m; r ⫽ 0.04, p ⫽ 0.89). These
data are therefore consistent with the ideathat 1-week-old adult-generated neurons
are insufficiently mature to be integrated
into hippocampal memory circuits (Kee etal., 2007b; Stone et al., 2010).
Together, these data suggest that stimulation increases the pool of functional
newborn neurons. An ANOVA, conducted on data from both experiments,
revealed a significant three-way Delay ⫻Thymidine Analog ⫻ Side interaction(F(1,30) ⫽ 4.79, p ⬍ 0.05). This supports
the conclusion that stimulation-induced
increases in contribution of adult-generated neurons to water-maze memory are
maturation dependent (they are observed
only in the 6 week group), as well as anatomically selective (only occurring on theside ipsilateral to the electrode site) andtemporally specific (corresponding to neurons generated 3–5 d after stimulation).
Furthermore, the temporal relationships
between stimulation, labeling (IdU and
CldU injections), and testing were equivalent in both experiments. Therefore, because IdU- and CldU-labeled cells wereequivalently aged at the time of testing, thesedata indicate that stimulation does not simply lower the threshold for activation(and/or Fos induction) of DGCs.
EC stimulation facilitates spatial memory
formation in a delay-dependent manner
We next asked whether stimulationinduced increases in neurogenesis would
facilitate water-maze learning in adultmice. To maximize production of newneurons, mice received bilateral (rather

Stone et al. • Entorhinal Cortex Stimulation Enhances Neurogenesis and Memory

Figure 6. Functional integration of stimulation-induced neurons into hippocampal memory networks. a, After unilateral stimulation, mice were injected with IdU during the period of increased proliferation and CldU once proliferation returned to baseline(n ⫽ 17). Mice were trained 6 weeks after CldU treatment, and spatial memory was assessed 4 weeks after the completion oftraining. b, Mice searched selectively in the probe test. A density plot for grouped data (left), with accompanying color scale,represents the number of visits per mouse per 5 ⫻ 5 cm area. Mice spent more time (right graph) searching the target zone (T)compared with other (O) zones. c, Representative confocal images of tissue after water-maze testing showing IdU ⫹, CldU ⫹, andFos ⫹ DGCs and a DAPI counterstained merge image with a representative IdU ⫹/Fos ⫹ colabeled cell (scale bar, 20 ␮m). d,
Numbers of IdU ⫹ and CldU ⫹ cells ipsilateral (I) versus contralateral (C) to stimulation site. e, Fos expression in the DG after probe
testing was similar on both sides. f, After the probe test, the probability of Fos ⫹ cells being XdU ⫹ ipsilateral (I) and contralateral(C) to stimulation ( y-axis notation denotes the conditional probability of a cell being XdU ⫹ given it is Fos ⫹, or P(XdU ⫹兩Fos ⫹)).
g, As the number of IdU ⫹ cells increases, so does their contribution to the population of activated neurons (i.e., P(IdU ⫹兩Fos ⫹)).
h, A separate group of mice underwent identical treatment except were trained 1 week after CldU treatment and had spatial
memory assessed 9 weeks after the completion of training (n ⫽ 15). i, Mice searched selectively in the probe test (left density plot),spending more time (right graph) searching the target zone compared with other zones. j, Numbers of IdU ⫹ and CldU ⫹ cells
ipsilateral (I) versus contralateral (C) to stimulation site. k, Fos expression in the DG after probe testing was similar on both sides.
l, The probability of Fos ⫹ cells being XdU ⫹ (or adult generated neurons) was equivalently low ipsilateral and contralateral tostimulation for IdU ⫹ and CldU ⫹ cells. m, The stimulation-induced increase in the availability of adult-generated neurons did not
produce a proportional increase in their contribution to the population of activated neurons, suggesting that neurons 1 week old at
the time of training are not functionally integrated. **p ⬍ 0.01.

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J. Neurosci., September 21, 2011 • 31(38):13469 –13484 • 13479

completion of training, spatial memory
was assessed in a probe test. In this test,
both stimulated and nonstimulated micesearched selectively (Fig. 7c). However, micein the stimulation group spent significantly more time in the target zone (Fig.
7d; unpaired t test, t(49) ⫽ 2.02, p ⬍ 0.05)
and crossed the former platform location
more frequently (Fig. 7e; unpaired t test,
t(49) ⫽ 3.83, p ⬍ 0.01) compared with
nonstimulated mice, indicating that
stimulation of the EC facilitated spatial
learning.
The beneficial effect of EC stimulation on spatial learning was detected in
the probe test but not in the training latency data. Because the adoption of either
localized/spatially precise (e.g., focal searching) or some nonlocalized/spatially imprecise (e.g., chaining) search strategies
may contribute to reduced escape latencies across training (Gallagher et al., 1993;
Lipp and Wolfer, 1998; Wolfer et al., 1998;
Clapcote and Roder, 2004), latency data
can be poor predictors of spatial learning(Clapcote and Roder, 2004). To address
whether stimulated and nonstimulated
mice adopted different search strategies
during training, we conducted a detailed
analysis of search paths. Using tracking
data and a criterion-based algorithm(Garthe et al., 2009), search paths were
objectively classified into eight mutually
exclusive categories (Fig. 7f ). At the beginning of training, thigmotaxis and random search strategies predominated inboth groups, accounting for ⬎75% of
paths (Fig. 7g). In contrast, by the end of
training, these spatially imprecise strategies were progressively replaced by more
localized/spatially precise strategies in2both stimulated (Friedman’s test, ␹(8)⫽73.89, p ⬍ 0.01) and control (Friedman’s
2
Figure 7. Stimulation-induced enhancement of spatial memory. a, At 6.5 weeks after bilateral stimulation (n ⫽ 25) or elec- test, ␹(8) ⫽ 66.66, p ⬍ 0.01) groups. Most
trode insertion only (n ⫽ 26), mice were trained in the water maze and spatial memory was assessed 1 h after the completion of strikingly, the overall frequencies oftraining. b, Latencies to reach the platform declined equivalently for stimulated (S) and nonstimulated (NS) mice during training. search strategies differed between groupsc, Mice searched selectively in the probe test. Density plots for grouped data, with accompanying color scale, represent the number (␹ 2 test of independence, ␹2 ⫽ 6.39, p ⬍(7)of visits per mouse per 5 ⫻ 5 cm area. d, e, Stimulated mice spent significantly more time searching the target zone (d) and 0.05). This difference was most apparent
crossing the target platform location (e) than nonstimulated mice. f, Examples of search strategies with corresponding color labels.
by an elevated prevalence of directg, Percentage of trials classified in each search strategy category across training days for stimulated (left graph) and nonstimulated
swims and focal searches in stimulated(right graph) mice. h, Percentage difference in the frequency of each search strategy in stimulated relative to nonstimulated mice,relative to nonstimulated mice (Fig. 7h).
indicating significantly greater use of direct swim and focal search strategies in stimulated mice. *p ⬍ 0.05, **p ⬍ 0.01.
Therefore, consistent with the probe
test, these analyses provide convergent
than unilateral) stimulation of the EC and were trained in theevidence that stimulation of the EC facilitated spatial learning
water maze 6.5 weeks later. Control mice were treated identicallyby promoting the adoption of localized/spatially precise
except that no current was delivered (Fig. 7a). This 6.5 week
search strategies.
stimulation-training delay ensures that additional neurons, proWhether this stimulation-induced facilitation of spatial learnduced as a consequence of stimulation, are sufficiently mature
ing is mediated by a stimulation-induced enhancement of adult(⬃6 weeks old) to contribute to spatial learning [previous experneurogenesis or some other mechanism is not clear. Consistent
iment (Kee et al., 2007b; Stone et al., 2010)]. During training,with previous studies (Kee et al., 2007b; Stone et al., 2010), we
latency to find the platform declined similarly in both groups
showed that 6-week-old, but not 1-week-old, adult-generated(Fig. 7b; Day ⫻ Stimulation ANOVA, significant main effect ofneurons are incorporated into DG circuits engaged during spatial
Day only, F(2,49) ⫽ 51.23, p ⬍ 0.01). Immediately after thelearning (Fig. 6). Therefore, the beneficial effects of EC stimula-

13480 • J. Neurosci., September 21, 2011 • 31(38):13469 –13484

Stone et al. • Entorhinal Cortex Stimulation Enhances Neurogenesis and Memorytion would be expected to have a delayed
onset if they are mediated by a neurogenic
mechanism. To test this, we next trained
mice 1.5 weeks, rather than 6.5 weeks, after stimulation of the EC (Fig. 8a). As before, both stimulated and control micerequired progressively less time to find theplatform during training, and there were
no differences between groups (Fig. 8b;
Day ⫻ Stimulation ANOVA, significant
main effect of Day only, F(2,49) ⫽ 64.14,
p ⬍ 0.01). Furthermore, both stimulated
and control mice progressed from predominantly nonlocalized/spatially imprecise strategies (e.g., thigmotaxis andrandom search) at the start of training topredominantly localized/spatially precise
strategies (e.g., direct swim, focal search,or directed search) at the completion oftraining (Fig. 8c; Friedman’s test for Stim2
ulated Group, ␹(8)⫽ 76.34, p ⬍ 0.01;
Friedman’s test for Nonstimulated Group,2
␹(8)⫽ 94.72, p ⬍ 0.01). In contrast to themice trained 6.5 weeks after stimulation,
our detailed analyses of swim paths during training revealed no differences in theprevalence of various search strategies between groups (Fig. 8d; ␹ 2 test of indepen2
dence, ␹(7)⫽ 3.30, p ⫽ 0.86). Consistent
with this, in the probe test 1 d after thecompletion of training, both stimulated
and control mice searched selectively (Fig.
8e), and the degree of selectivity did not
differ between groups: both stimulated
and nonstimulated mice spent equivalent
time searching the target zone (Fig. 8f; unpaired t test, t(49) ⫽ 1.11, p ⫽ 0.27) andcrossed the former platform location withsimilar frequency (Fig. 8g; unpaired t test,
t(49) ⫽ 0.60, p ⫽ 0.55). Therefore, spatial
learning is not facilitated when training
takes place 1.5 weeks after stimulation, Figure 8. Specificity of stimulation-induced enhancement of spatial memory. a, At 1.5 weeks after bilateral stimulation (n ⫽suggesting that the beneficial effects of 25) or electrode insertion only (n ⫽ 26), mice were trained in the water maze and spatial memory was assessed 1 h after thecompletion of training. b, Latencies to reach the platform declined equivalently for stimulated (S) and nonstimulated (NS) micestimulation are delayed in their onset.
These data strengthen the causal link during the training period. c, Percentage of trials classified in each search strategy category across training days for stimulated (leftbetween stimulation-induced increases in graph) and nonstimulated (right graph) mice. d, Percentage difference in the frequency of each search strategy in stimulated
adult neurogenesis and facilitated spatial relative to nonstimulated mice, indicating minimal variability between groups. e, Mice searched selectively in the probe test.
learning. Six-week-old (but not 1-week- Density plots for grouped data, with accompanying color scale, represent the number of visits per mouse per 5 ⫻ 5 cm area. f, g,
Percentage time searching the target zone (T) compared with other (O) zones (f ) and target platform crossings (T) compared withold) adult-generated neurons are engaged
other platform locations (O) (g) for stimulated (S) versus nonstimulated (NS) mice in the probe test. h, Separate groups of mice
during spatial learning. Stimulation in- received bilateral stimulation (n ⫽ 24) or electrode insertion only (n ⫽ 24) 1 d after the completion of water-maze training, andcreases neurogenesis, and the facilitatory spatial memory was assessed 1.5 weeks after surgery. i, Latencies to reach the platform declined equivalently for both groups
effects of stimulation-induced changes in during the training period. j, Percentage of trials classified in each search strategy category across training days for stimulated (leftneurogenesis do not emerge until 6.5 graph) and nonstimulated (right graph) mice. k, Percentage difference in the frequency of each search strategy in stimulated
weeks after stimulation. As an additional relative to nonstimulated mice, indicating minimal variability between groups. l, Mice searched selectively in the probe test.
test of this idea, an additional group of Density plots for grouped data, with accompanying color scale, represent the number of visits per mouse per 5 ⫻ 5 cm area. m, n,
mice received stimulation after (rather Both groups spent more time searching the target zone (m) and crossing the target platform location (n) compared with otherthan before) training in the water maze zones and platform locations, respectively; however, there were no differences in these measures between groups.
(Fig. 8h). It is unlikely that new neurons
platform (Fig. 8i; Day ⫻ Stimulation ANOVA, significant main
would be retroactively integrated into dentate circuits supporting
effect of Day only, F(2,46) ⫽ 69.96, p ⬍ 0.01) and progressed fromspatial memory. Therefore, it is unlikely that new neurons, genpredominantly nonlocalized/spatially imprecise to localized/spaerated as a consequence of stimulation, would contribute to spatially precise strategies (Fig. 8j; Friedman’s test for Stimulated
tial learning in this experiment. As expected, during training,
2
Group, ␹(8)both groups of mice required progressively less time to locate the⫽ 40.43, p ⬍ 0.01; Friedman’s test for Nonstimulated

Stone et al. • Entorhinal Cortex Stimulation Enhances Neurogenesis and Memory

J. Neurosci., September 21, 2011 • 31(38):13469 –13484 • 13481

Figure 9. Suppressing the stimulation-induced increase in neurogenesis blocks spatial memory enhancement. a, At 1 d after the completion of V or TMZ injections, four groups of mice underwent
bilateral stimulation (S) or electrode insertion only (NS) (V-S, n ⫽ 24; V-NS, n ⫽ 17; TMZ-S, n ⫽ 24; TMZ-NS, n ⫽ 17). Six and a half weeks after surgery, all mice were trained in the water maze,and spatial memory was assessed 1 h after the completion of training. Subsets of these mice received BrdU injections either during an early poststimulation period (V-S, n ⫽ 8; V-NS, n ⫽ 8; TMZ-S,n ⫽ 5; TMZ-NS, n ⫽ 5) or after the completion of probe testing (V-S, n ⫽ 5; V-NS, n ⫽ 7; TMZ-S, n ⫽ 7; TMZ-NS, n ⫽ 5). b, BrdU ⫹ cells (green) labeled during the early poststimulation period(NeuN counterstain; scale bar, 50 ␮m). c, BrdU incorporation after labeling during the early poststimulation period. The stimulation-induced increase in proliferation was blocked by TMZ treatment.
d, Similar proportions of BrdU ⫹ cells were NeuN ⫹ in each group when mice received BrdU injections during the early poststimulation period. e, Latencies to reach the platform declined equivalently
for all groups of mice during the training period. f, Density plots for grouped data, with accompanying color scale, represent the number of visits per mouse per 5 ⫻ 5 cm area. f, g, V-S mice spentsignificantly more time searching the target (T) zone compared with all other groups. h, BrdU incorporation after behavioral tests was similar across groups. i, Similar proportions of these BrdUⴙ cells
costained for NeuN across groups. *p ⬍ 0.05, **p ⬍ 0.01.
2Group, ␹(8)⫽ 46.02, p ⬍ 0.01) with equivalent tendency (Fig. 8k;22
␹ test of independence, ␹(7)⫽ 1.57, p ⫽ 0.98). Similarly, in thesubsequent probe test, both groups searched selectively in theregion of the pool that formerly contained the platform (Fig. 8l ).
Because the degree of selectivity did not differ between groups(Fig. 8m,n; unpaired t test for Zones, t(46) ⫽ 0.32, p ⫽ 0.75;
unpaired t test for Platform Crossings, t(46) ⫽ 0.19, p ⫽ 0.85),
these results exclude the possibility that stimulation of the EC
nonspecifically facilitates memory retrieval. Moreover, they also
indicate that stimulation does not disrupt the expression of apreviously acquired memory.

Facilitation of spatial memory is prevented by blocking
adult neurogenesis
To more directly evaluate whether EC stimulation facilitates theformation of spatial memory via a neurogenic mechanism, wenext examined the impact of inhibiting neurogenesis using the
DNA-alkylating agent TMZ (Garthe et al., 2009). In this experiment, stimulated or nonstimulated control mice were pretreatedwith V or TMZ. Six and a half weeks later, they were trained in thewater maze (three trials per d for 3 d), and then spatial memory
was assessed in a probe test 1 h after the completion of training(Fig. 9a). To assess neurogenesis at different poststimulation time
points, mice were additionally treated with BrdU either 3–5 dpostoperatively (i.e., a time point corresponding to the peak instimulation-induced changes in proliferation) or after watermaze testing (a time point when proliferation rates would be
expected to have returned to baseline or ⬃7 weeks postoperatively). In mice treated with BrdU soon after training, BrdU incorporation differed between groups (Fig. 9b,c; ANOVA Groupeffect, F(3,22) ⫽ 14.91, p ⬍ 0.001), indicating that proliferation
was altered at this early poststimulation time point. Most notably, in V-treated mice, EC stimulation increased proliferation, as
we had observed previously (Duncan’s post hoc test, p ⬍ 0.01),
and this stimulation-induced increase was attenuated by TMZ
treatment (TMZ-S ⬍ V-S, Duncan’s post hoc test, p ⬍ 0.01).
Additionally, TMZ treatment reduced proliferation by ⬃45% in
nonstimulated mice (Duncan’s post hoc test, p ⬍ 0.05), as expected. There were no group differences in the proportion of
BrdU-labeled cells expressing the neuronal marker NeuN (Fig.
9d; ANOVA Group effect, F(3,22) ⫽ 0.25, p ⫽ 0.86), indicatingthat these treatment combinations did not alter the fate of newly
generated cells.
During training, latency to find the platform declined similarly in all groups (Fig. 9e; Day ⫻ Group ANOVA, significant
main effect of Day only, F(2,156) ⫽ 27.54, p ⬍ 0.001). However, inthe probe test after training, the V-treated, stimulated group outperformed all other groups, spending more time in the target
zone (Fig. 9f,g; ANOVA on target zone data, Group effect, F(3,78) ⫽4.69, p ⬍ 0.01, Duncan’s post hoc tests, p ⬍ 0.05). These data there-13482 • J. Neurosci., September 21, 2011 • 31(38):13469 –13484

fore replicate our previous finding that EC stimulation facilitates
spatial memory formation (V-S group ⬎ V-NS group) and, mostimportantly, show that this enhancement is prevented when thestimulation-induced enhancement of neurogenesis is blocked.
Both TMZ and stimulation only transiently altered neurogenesis levels because BrdU incorporation was equivalent acrossgroups in mice treated with BrdU after behavioral testing (Fig.
9h; ANOVA Group effect, F(3,20) ⫽ 0.13, p ⫽ 0.94), and there
were no group differences in the proportion of BrdU-labeledcells expressing the neuronal marker NeuN (Fig. 9i; ANOVAGroup effect, F(3,20) ⫽ 1.06, p ⫽ 0.39).

Discussion
Although DBS targeting hippocampal afferents may slow or arrest cognitive decline (Laxton et al., 2010), the underlying mechanisms of action are poorly understood. Using a mouse model,
we provide four lines of evidence that such pro-cognitive effects
are mediated, at least in part, by activity-dependent promotion ofhippocampal neurogenesis. First, using parameters analogous toclinical high-frequency DBS, we show that targeted stimulationof EC in adult mice produces an anatomically and temporally
specific increase in DGC production. Relative to cells produced
under basal conditions, stimulation-induced cells exhibit comparable rates of neuronal differentiation and long-term survival.
Second, new neurons produced as a consequence of EC stimulation assume functional roles in hippocampal circuits. Relative tocells produced under basal conditions, they show comparable
localization in the DG, mature normally, and integrate into hippocampal networks engaged by spatial memory in a maturationdependent manner. Third, acute, bilateral stimulation of the EC
facilitated spatial memory formation. Facilitation only emerged
several weeks after stimulation and not if stimulation occurred⬃1 week before or after training. This time course tracks exactly
the maturation-dependent time course of integration of adultgenerated neurons into spatial memory circuits. Fourth, facilitation of spatial memory formation was prevented by blocking
neurogenesis, suggesting that the pro-cognitive effects of EC stimulation are most likely mediated by a neurogenic mechanism.
We found that stimulation for 1 h was sufficient to promote
proliferation in the DG. The pro-proliferative effects of EC stimulation had distinct temporal and anatomical profiles. Similar toelectroconvulsive shock (Ma et al., 2009), induced seizures (Parent et al., 1997; Jiang et al., 2003), and targeted brain stimulation(Bruel-Jungerman et al., 2006; Toda et al., 2008), increased proliferation occurred in a delayed manner and peaked 3–5 d after
stimulation before returning to baseline. Furthermore, after unilateral stimulation, increased proliferation was limited to the sideipsilateral to the electrode site. This is consistent with previous
studies showing that the perforant path projection from layer II
medial/lateral EC neurons is almost exclusively ipsilateral in themouse (van Groen et al., 2002, 2003; Amaral and Lavenex, 2007).
Similar to previous studies, we found that stimulation did notalter rates of neuronal differentiation or apoptosis, suggestingthat these effects are most likely mediated instead by increasing
the proliferative activity of neural progenitor cells in the subgranular zone (Bruel-Jungerman et al., 2006; Toda et al., 2008;
Kitamura et al., 2010; Encinas et al., 2011). The subgranular zone
contains cells with varying degrees of multipotency and capacities
for self-renewal and proliferation. The most proliferative of these
are type 2 progenitor cells, whose progeny progressively differentiate into DGCs. Stimulation of another limbic target, the anterior thalamic nucleus, promotes adult neurogenesis in thesubgranular zone by increasing the number and mitotic activity

Stone et al. • Entorhinal Cortex Stimulation Enhances Neurogenesis and Memoryof type 2 progenitor cells (Encinas et al., 2011), leading to increased DGC production (Toda et al., 2008; Encinas et al., 2011).
Similarly, other pro-neurogenic stimuli, such as seizures and electroconvulsive shock, primarily increase neurogenesis through increased progenitor cell proliferation, suggesting shared underlying
mechanisms (Parent and Murphy, 2008; Zhao et al., 2008).
Under basal conditions, granule cells generated during adulthood take several weeks to fully mature and establish functional
afferent and efferent connections (Zhao et al., 2006; Toni et al.,2007, 2008). Using a retroviral strategy to label adult-generated
granule cells, our data indicate that neuronal maturation follows
an equivalent time course after unilateral EC stimulation: one
week after infection, retrovirally labeled cells typically had short,aspiny, dendritic processes and axons that had not yet reached theCA3 region, whereas 6 weeks after infection, retrovirally labeledcells had mature neuronal morphology, with spiny, highly arborized dendritic processes extending into the molecular layer
and axons extending into CA3. Because labeled neurons had
similar properties both ipsilateral and contralateral to the stimulation site, these data suggest that neurons produced as a consequence of stimulation acquire normal morphology and likely
establish normal afferent and efferent connections. Other conditions, such as epileptic activity (Parent et al., 1997; Jessberger etal., 2007), may also lead to a sustained increase in adult hippocampal neurogenesis. However, after epileptic activity, newly
generated neurons develop abnormal morphology, and many become ectopically located in the hilus. In contrast, we found noevidence for abnormal morphology or ectopic localization after
acute stimulation, suggesting that the low-current intensity stimulation that we used does not promote the aberrant integration of
adult-generated neurons into hippocampal circuits.
Previous studies have shown that, once sufficiently mature,adult-generated neurons are activated during memory formation
and/or expression (Kee et al., 2007b; Tashiro et al., 2007; Trouche
et al., 2009; Stone et al., 2010). In our experiments, EC stimulation nearly doubled levels of neurogenesis, and there was a corresponding increase in the numbers of adult-generated neurons
in the pool of granule cells activated by the expression of a watermaze memory. This suggests that stimulation-induced neurons
become functionally integrated into hippocampal networks supporting spatial memory. Importantly, this increase was specific toneurons generated 3–5 d after stimulation and limited to the sideipsilateral to the stimulation site and only when stimulation preceded training by 6.5 weeks. This finding is consistent with previous studies showing that integration of newborn DGCs into
hippocampal circuits supporting water-maze memory is regulated by cell age, with newborn DGCs not maximally contributing until they are 5 weeks or older (Kee et al., 2007b; Stone et al.,2010). This delayed time course for functional integration follows
the establishment and gradual maturation of excitatory connections of newborn DGCs (Piatti et al., 2006; Zhao et al., 2008).
Most strikingly, bilateral stimulation of the EC facilitated theformation of a hippocampus-dependent water-maze memory. In
the probe test after training, stimulated mice searched more selectively compared with nonstimulated controls. Detailed analyses of swim paths during training revealed that stimulated mice
were more likely to use localized/spatially precise search strategies (e.g., direct swim, focal search) and that the increased frequency of these more effective strategies very likely accounts forthe improved spatial memory formation. In these types of experiments, a key question is whether improved memory formation iscausally related to changes in neurogenesis. For example, although environmental enrichment both promotes adult neuro-

Stone et al. • Entorhinal Cortex Stimulation Enhances Neurogenesis and Memory

genesis and facilitates memory formation, its memory effects are
independent of those on neurogenesis (Meshi et al., 2006). In our
experiments, stimulation of the EC may produce a range of othereffects that, in principle, could contribute to improved spatial
memory formation. These might include effects on developmentally generated hippocampal neurons (e.g., dendritic remodeling) and/or on the differentiation and maturation of existing
adult-generated granule cells. However, memory facilitation was
fully blocked by pharmacological blockade of the stimulationinduced increase in neurogenesis. Because TMZ would predominantly affect proliferation (and not postmitotic cells), thisexperiment reduces the likelihood that these non-neurogenic
mechanisms contribute to the pro-cognitive effects of EC stimulation. One related concern is that TMZ treatment may impact
general health. However, TMZ treatment did not impair spatial
memory formation in nonstimulated mice. Moreover, even more
prolonged TMZ treatment does not affect weight, induce neuroinflammation, or alter general behavioral or hematological profiles in mice (Garthe et al., 2009), and so it seems unlikely thatthese potential off-target effects confound our results.
Our experiments suggest that promotion of adult neurogenesis is one plausible mechanism by which DBS might exert procognitive effects. In translating these findings to clinical settings,
several issues are worth considering. First, mechanisms underlying the pro-cognitive effects of DBS likely include, but need not
be limited to, activity-dependent promotion of adult neurogenesis. For example, stimulation may induce neurotransmitter release and local or transsynaptic modulation of neural activity at
the cellular level (Kringelbach et al., 2007), as well as restore basal
activity levels within dysregulated brain regions at the circuit level(Mayberg et al., 2005; Laxton et al., 2010). Such effects might act
in concert with stimulation-induced changes in neurogenesis topromote cognitive recovery. DBS of hippocampal afferents may
additionally have immediate effects on memory [e.g., vivid autobiographical recall seen during initiation of stimulation in someof our patients (Hamani et al., 2008; Laxton et al., 2010)], andthese are unlikely to be mediated by a neurogenic mechanism.
Second, pro-neurogenic effects of stimulation are not limited to
the EC. For example, stimulation of other limbic targets increases
adult neurogenesis (Derrick et al., 2000; Toda et al., 2008; Encinas
et al., 2011). Whether such stimulation has beneficial effects on
cognitive function remains to be determined. Indeed, stimulation of the anterior thalamic nucleus may impair, rather thanfacilitate, cognitive function in rats (Hamani et al., 2010), andthese effects are consistent with memory impairments reported
in some DBS-implanted epilepsy patients (Fisher et al., 2010).
Third, although we used short-duration stimulation in our experiments, in clinical settings DBS typically involves chronic intermittent or continuous stimulation. The main advantage ofusing acute stimulation was that it allowed us to isolate effects ofstimulation-induced neurogenesis on behavior. Although moresustained stimulation might bring greater cognitive benefit, it isimportant to recognize that chronic EC stimulation, albeit likely
at higher intensity than used here, carries a greater risk of toxicity(Sloviter et al., 1996).

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