NeuroImage 125 (2016) 422–427

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NeuroImage
journal homepage: www.elsevier.com/locate/ynimg

Deep brain stimulation of the ventromedial prefrontal cortex causes
reorganization of neuronal processes and vasculature
M. Mallar Chakravarty a,b,c,⁎,1, Clement Hamani e,f,1, Alonso Martinez-Canabal g, Jacob Ellegood h,
Christine Laliberté h, José N. Nobrega d,f, Tejas Sankar e,i, Andres M. Lozano e,
Paul W. Frankland g,k,l,m, Jason P. Lerch g,h,ja

Cerebral Imaging Centre, Douglas Mental Health University Institute, Canada
Department of Psychiatry, McGill University, CanadacDepartment of Biomedical Engineering, McGill University, CanadadDepartment of Psychiatry, University of Toronto, CanadaeDivision of Neurosurgery, Toronto Western Hospital, CanadafBehavioural Neurobiology Laboratory, Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, CanadagProgram in Neuroscience and Mental Health, The Hospital for Sick Children, CanadahMouse Imaging Centre (MICe), The Hospital for Sick Children, CanadaiDivision of Neurosurgery, University of Alberta, CanadajDepartment of Medical Biophysics, University of Toronto, CanadakDepartment of Psychology, University of Toronto, Toronto, CanadalDepartment of Physiology, University of Toronto, Toronto, CanadamInstitute of Medical Science, University of Toronto, Toronto, Canadab

a r t i c l e

i n f oArticle history:
Received 27 March 2015
Accepted 18 October 2015
Available online 23 October 2015a b s t r a c t
Background: Chronic high-frequency electrical deep brain stimulation (DBS) of the subcallosal cingulate region iscurrently being investigated clinically as a therapy for treatment of refractory depression. Experimental DBS of
the homologous region, the ventromedial prefrontal cortex (VMPFC), in rodent models has previously demonstrated anti-depressant-like effects. Our goal was to determine if structural remodeling accompanies the alterations ofbrain function previously observed as a result of chronic DBS.
Methods: Here we applied 6 h of high-frequency bilateral VMPFC DBS daily to 8 9-week old C57Bl/6 mice for 5 days.
We investigated the “micro-lesion” effect by using a sham stimulation group (8 mice) and a control group (8 micewith a hole drilled into the skull only). Whole brain anatomy was investigated post-mortem using high-resolution
magnetic resonance imaging and areas demonstrating volumetric expansion were further investigated using histology and immunohistochemistry.
Results: The DBS group demonstrated bilateral increases in whole hippocampus and the left thalamus volume compared to both sham and control groups. Local hippocampal and thalamic volume increases were also observed at thevoxel-level; however these increases were observed in both DBS and sham groups. Follow-up immunohistochemistry in the hippocampus revealed DBS increased blood vessel size and synaptic density relative to the control groupwhereas the sham group demonstrated increased astrocyte size.
Conclusions: Our work demonstrates that DBS not only works by altering function with neural circuits, but also by
structurally altering circuits at the cellular level. Neuroplastic alterations may play a role in mediating the clinical efﬁcacy of DBS therapy.
© 2015 Elsevier Inc. All rights reserved.

Introduction
High-frequency deep brain stimulation (DBS) delivered using aneurosurgically implanted electrode has been applied with great clinical⁎ Corresponding author at: Cerebral Imaging Centre, Douglas Mental Health University
Institute, 6875 LaSalle Boulevard, Verdun, QC H4H 1R3, Canada.
E-mail address: mallar@cobralab.ca (M.M. Chakravarty).
1
These two authors contributed equally to this work.

http://dx.doi.org/10.1016/j.neuroimage.2015.10.049
1053-8119/© 2015 Elsevier Inc. All rights reserved.

efﬁcacy in the treatment of Parkinson's disease and other movement
disorders (Limousin et al., 1995). Given the success of this treatment
for movement disorders, the use of chronic DBS has been proposed asa treatment for several other neuropsychiatric conditions as a meansof compensating for malfunctioning brain circuitry (Lozano and
Lipsman, 2013). Recently, the subcallosal cingulate region has beenused as a DBS target in treatment refractory depression (TRD) (Lozano
et al., 2008; Mayberg et al., 2005), with patients receiving the therapy
demonstrating sustained anti-depressant response in 3–6 year follow-

M.M. Chakravarty et al. / NeuroImage 125 (2016) 422–427

up open label studies (Kennedy et al., 2011) in a clinical population at
high-risk for suicide (Conwell and Brent, 1995). In patients with TRD,
local and remote changes in brain function in response to the focal application of subcallosal DBS have been previously demonstrated andmay account, in part, for the therapeutic mechanism of action of DBS
in depression (Lozano et al., 2008; Mayberg et al., 2005). Experimental
studies involving stimulation of the ventromedial prefrontal cortex(VMPFC; the rodent homologue of the subcallosal cingulate region) innormal wild-type rats demonstrate the alleviation of depressive-like behaviors due to being subjected to a forced swim test (Hamani et al.,2010, 2012). Recent ﬁndings from our group show that in addition tofunctional remodeling, DBS applied in the context of neuropsychiatricdisorders may mediate clinical efﬁcacy through neuroanatomical remodeling as well. For example, DBS applied to key nodes in the memory
circuit of rodents (Laxton et al., 2010) (such as the entorhinal cortex andthe anterior nucleus of the thalamus) induces the generation of newneurons that, once mature, integrate into hippocampal memory circuits
responsible for the maintenance of memory (Hamani et al., 2011; Stoneet al., 2011). There is mounting evidence that neuroanatomical reorganization is also occurring in patient populations being treated with
DBS. In a recent clinical ﬁnding, two Alzheimer's disease patients demonstrated hippocampal growth and improvement in their clinical presentation in response to chronic administration of DBS to the fornix(Sankar et al., 2015).
However, in the context of TRD, it is unclear as to whether or not thesechanges in brain function and behavior are accompanied or mediated byneuroanatomical reorganization, and if so, in what part of the associatednetwork remodeling occurs. There is also recent evidence that the
microlesion caused by the implantation of the DBS electrode itself may account for some improvement in symptomatology. We do note, however,that the use of lesion-otomies has been previously proposed in the treatment of major depression, often with mixed results. These include lesions
throughout the cingulate, the internal capsule, and the tracts adjacent tothe caudate nucleus (Shah et al., 2008); therefore there may be a neuroanatomical basis for remodeling observed due to microlesions and we
speculate that neuroanatomical remodeling may be further modulated
by chronic deep brain stimulation (possibly leading to therapeutic efﬁcacy). While the microlesion phenomenon has been receiving some attention in the Parkinson's disease literature (Borden et al., 2014; Le Goffet al., 2014; Maltete et al., 2008, 2009), it has been relatively
understudied.
Given the emerging interest in the subcallosal cingulate with respect
to other neuropsychiatric disorders, such as treatment-refractory anorexia nervosa (Lipsman et al., 2013), it is critical to determine whether and towhat extent neuroanatomical remodeling, and more speciﬁcally, if changes in neuronal processes, glial cells, and vasculature occur in response to
DBS of this region. In this manuscript, we tested this open question by applying DBS to the ventromedial prefrontal cortex (VMPFC; the rodent homologue to the human subcallosal cingulate (Hamani et al., 2010, 2012))
of 8 9-week-old male C57Bl/6 mice. We used whole-brain highresolution magnetic resonance imaging (MRI) data to analyze the neuroanatomical effects on the stimulated groups. To test the micro-lesion effect we also analyzed the neuroanatomy of a group receiving shamstimulation (i.e., stimulator implanted but no active stimulation
received). Volumetric changes observed using MRI analysis were thenused to guide histological and immunohistochemical analyses.

423

Kopf Instruments; Tujunga, CA, USA). Electrodes with a diameter of
125 μm and a 0.5 mm2 of exposed surface were connected to a plastic
pedestal (Plastics One; Roanoke, VA, USA) and bilaterally implanted inthe VMPFC (anteroposterior +1.7, lateral ±0.2, depth 1.2 mm) andused as cathodes (Franklin and Paxinos, 2004). A screw implanted overthe somatosensory cortex was used as the anode. Two additional screws
were attached to the skull for better securing the cap in place. Electrodes
and screws were ﬁxed to the skull with dental acrylic cement. A week
after surgery, the DBS group received stimulation for 6 h a day over ﬁve
days (similar to the design previously used from our group in the studyof rats (Hamani et al., 2010, 2012); stimulation parameters: 50 μA,
130 Hz, 90 μs pulse width). These settings were selected as they havebeen previously effective in improving memory performance in the Morris water maze (Stone et al., 2011) and approximate those used in mostclinical applications of DBS if the charge density is taken into account.
To test the inﬂuence of the so-called “micro-lesion effect”, where
symptom improvement is noted shortly after electrode implantation
and before DBS has been delivered or optimized (Lozano et al., 2008;
Mayberg et al., 2005; Cersosimo et al., 2009; Granziera et al., 2008),
we studied an additional group (n = 8) receiving sham stimulation(i.e., where the stimulator is implanted and not turned on). A third control group of 8 mice had holes drilled in the skull but no electrodes implanted (total: 24 mice total, all same background strain, males, and age
as the DBS group). The control group was handled over 5 days in orderto mimic the handling of both sham and DBS groups.
Magnetic resonance imaging (MRI)Three days after the end of stimulation mice were perfused throughthe left cardiac ventricle with 30 mL of phosphate-buffered saline (PBS)(pH 7.4) containing 2 mM of ProHance® (Bracco Diagnostics Inc.,
Princeton, NJ) at room temperature (25 °C). This was followed by infusion with 30 mL of 4% paraformaldehyde (PFA) and 2 mM of ProHance®
(Bracco Diagnostics Inc., Princeton, NJ) in PBS at room temperature. Following perfusion, the heads were removed along with the skin, lower
jaw, ears and the cartilaginous nose tip. The remaining skull structurescontaining the brain were allowed to postﬁx in 4% PFA and 2 mM of
ProHance® at 4 °C for 12 h and was stored in this fashion for 72 hprior to imaging. A multi-channel 7.0 T MRI scanner (Agilent, Palo
Alto, CA) was used to acquire anatomical images of brains within skulls(Nieman et al., 2007). Prior to imaging, the samples were removed fromthe contrast agent solution, blotted and placed into 13-mm-diameterplastic tubes ﬁlled with a proton-free susceptibility-matching ﬂuid
(Fluorinert FC-77, 3 M Corp., St. Paul, MN). An array of 16 custom built
solenoid coils was used for high-throughput scanning of the specimens
using a T2-weighted 3D Fast Spin Echo (FSE) sequence, with TR =2000 ms, echo train length = 6, TEeff = 42 ms, FOV of
25 mm × 28 mm × 14 mm, and a matrix size of 450 × 504 × 250. This
yields an isotropic (3D) resolution of 56 μm. In the ﬁrst phase encode dimension, consecutive k-space lines were assigned to alternating echoes
to move discontinuity related ghosting artifacts to the edges of the FOV.
This sequence involves oversampling in the phase encoding direction bya factor of 2 to avoid the interference of these artifacts. This FOV direction was subsequently cropped to 14 mm after reconstruction. Total imaging time was ~12 h.
Image processing for MRI data

Materials and methods
Surgeries, electrode implantation and deep brain stimulation
To test how DBS may impact neuroanatomical networks and brainwide neuroanatomical remodeling we applied DBS to 8 C57Bl/6 mice(~25 g; Charles River, Wilmington, Massachusetts, USA; all males;
9 weeks old). Mice were anesthetized with ketamine/xylazine (75/
10 mg/kg i.p.) and had their heads ﬁxed to a stereotaxic frame (David

Anatomical changes accompanying DBS were estimated using
deformation-based analysis of the T2-weighted MRI data (as describedin previous publications from our group (Lerch et al., 2008)). All image
processing was carried out with images in minc format using the minc
suite of software tools (http://www.bic.mni.mcgill.ca/ServicesSoftware/
ServicesSoftwareMincToolKit). All MRI volumes were rigidly rotated andtranslated (3 rotations and 3 translations) to match an initial atlas. All possible pair-wise 12-parameter transformations (3 rotations, translations,424

M.M. Chakravarty et al. / NeuroImage 125 (2016) 422–427

scales and shears) (Collins et al., 1994) were estimated and an average
linear transformation was calculated for each image, thus effectively scaling each brain to the average size of the population. After applying the average transformation, MRI volumes were averaged in order to create aﬁrst population-based model. A multi-generation, multi-resolution ﬁtting
strategy was then initialized where each brain is nonlinearly registeredusing the ANTs algorithm (Avants et al., 2008), modiﬁed to work with
MINC data (https://github.com/vfonov/ANTs) and for the mice brain(van Eede et al., 2013). Each mouse brain was nonlinearly registered to
the 12-parameter population atlas, and subsequently to the atlas of theprevious nonlinear registration. Three generations were estimated, as
per previous work from our group (van Eede et al., 2013). The registration
parameters at each stage have are detailed in Supplementary Table 1. The
ﬁnal deformation ﬁelds map each T2-weighted volume to the average ofthe entire population based on the minimum deformation required tomap each subject to the average of the group (i.e., the so-called minimum
deformation template). Using these ﬁnal deformations, we customized anatlas containing 62 neuroanatomical areas to each of the mice in the study
to enable the estimation of structure volume (Dorr et al., 2008). The logtransformed Jacobian determinant (Chung et al., 2001), a measure of local
expansion and contraction, was estimated at each node in the ﬁnal deformation ﬁelds. Statistical analysis was carried out using a voxel-by-voxel
general linear model included in the RMINC package (https://wiki.
phenogenomics.ca/display/MICePub/RMINC) and all results werecorrected for multiple comparisons using the False Discovery Rate
(Genovese et al., 2002). All mice were analyzed through the developmentof a single group-wise average to enable direct comparisons between all
through of the groups. We have previously used this method to estimate
neuroanatomical differences in response to different maze training strategies learned over 5 days (Lerch et al., 2011), a week of motor skills training (Scholz et al., 2015), or 24 h of environmental enrichment (Scholz
et al., 2015), demonstrating that this method is sensitive to subtle neuroanatomical differences between mice.
Histological/immunohistochemical processing and imaging
50 μm sections were cut in a cryostat (Leica CM1850; Wetzlar,
Germany) from regions of interest. Sucrose 30% in PBS was used to submerge the brains before slicing. The brain sections were stored in thefreezer using 10% ethylene glycol and 40% glycerol in PBS.
Each sixth section was placed in a well with antifreezer solution (50%glycerol [Sigma; St. Louis, MO, USA], 10% ethylene glycol [Sigma] and kept
at −20 °C until use. Sections were thoroughly washed and placed in citrate buffer (Sigma) pH 6.0 at 95 C during 1 h. After washed, the sections
were blocked for peroxidase activity in 0.3% H2O2 during 30 min, later
were blocked with normal goat serum 3%, bovine serum albumin 1.5%
and Tx100 0.3% during 2 h. Sections were immediately transfer to the primary antibody. With all the primary antibodies used, sections were incubated at room temperature ON. After being thoroughly washed, someprimary antibodies were detected directly with appropriate secondary
ﬂuorescent antibodies, mouse glial ﬁbrillary acidic protein (GFAP; cell signaling, Danvers, MA, USA) was detected using antimouse-Alexa 568(Invitrogen, Carlsbad, CA, USA); rabbit polyclonal anti-collagen IV(1:1000; Chemicon, Temecula, CA, USA) was detected using anti-rabbit
Alexa 488. Rabbit Synaptophysin (Abcam, Cambridge, England, United
Kingdom) was detected using biotynilated anti-rabbit (Jackson),followed by avidin biotin complex (Vector; Burlingame, CA, USA) anddeveloped using bio-tyramide signal ampliﬁcation kit (Invitrogen)and streptavidin-horseradish peroxidase-568 (Invitrogen). After color
developing, all sections were placed in 4′,6-diamidino-2-phenylindole
solution (DAPI; Sigma) during 20 min, washed again, mounted andcoverslipped with permaﬂuor (Thermo Scientiﬁc; Waltham, MA, USA).
For each antibody, slices were selected at 300 μm. After a series was
selected for collagen, GFAP, and DAPI, the next series was selected forsynaptophysin. For every antibody tested, we performed previous
probes of staining without the antibody where no staining was shown

besides the usual background commonly observed in ﬂuorescentprobes upon brain tissue. We also examined that the anatomic localization of the signal corresponded with previous literature. Finally, imagesof the structures were taken with 10 × and 20 × objectives ofa microscope Olympus BX61 (Center Valley, PA, USA), with a Reiga
camera 1100.
Histological/immunohistochemical image analysis
Digitized sections were analyzed using the open-source CellProﬁler
software (Carpenter et al., 2006). The following CellProﬁler modules
and parameters were used for GFAP and collagen: 1) unmix colors;2) invert each separate color; 3) identify primary objects (astrocytes)
with a typical diameter of 4–90 pixels using the Otsu method andwith intensity to separate clumped objects; 4) identify primary objects(vessels) with a typical diameter of 10–400 pixels using the Otsu method and with intensity to separate clumped objects. For synaptophysin:
1) correct illumination using splines with 4 spline points; 2) convert
to grayscale; 3) identify primary objects (DAPI) with a typical diameterof 6–30 pixels using the Otsu method; 4) mask the input image with theidentiﬁed nuclei from the DAPI stain retaining everything outside of thenuclei; 5) rescale intensity to be between 0 and 1; and 6) measure
image intensity.
Statistical analysis of histological data
All statistical analyses were carried out in R (http://www.r-project.
org/). Comparisons between DBS, sham, and control groups were carried out using mixed-model regression as it permits the inclusion ofmultiple measurements per mouse (Pinheiro and Bates, 2000). Correlations between the histological data and the Jacobian determinant were
performed using the mean value from the histological analysis
described above. In cases where greater than three measurements fora single structure were available, a trimmed mean (removing the
maximum and minimum values) was used.
Results
All images were successfully processed through the automatedimage processing pipeline detailed in Section 2.3. It is clear that thepipeline is able to handle discrepant morphology created by the DBS
electrode (see Supplementary Materials Fig. 1). Since we expected
artefactual volumetric expansion in the vicinity of the site where theelectrode was inserted, we instead focused our analysis on volumetric
expansions observed distal to this region.
After correcting for multiple comparisons for 62 structures using theFalse Discovery Rate (FDR) (Genovese et al., 2002), bilateral hippocampal
volume was larger in the DBS group in comparison to both the sham (5%FDR, t = 5.3) and control groups (10% FDR, t = 3.8; see Fig. 1A, i). The volume of the left thalamus was also larger in the DBS group in comparison
to both the sham (5% FDR, t = 4.9) and control (10% FDR, t = 3.6) groups.
After images were analyzed for volumetric expansion at the voxel-level,
we observed volumetric expansions in four speciﬁc locations that survived 5% FDR correction when comparing DBS and control groups: inthe anterior portion of the left hippocampus (t = 3.3), bilaterally in the
body of the hippocampus (t = 3.0 and t = 2.8; left and right respectively; see Fig. 1B, i), and in the anterior portion of the left thalamus (t =2.1; see Fig. 1B, ii) in comparison to the control mice. Surprisingly, thesham group also demonstrated volumetric increase relative to controls
in the same regions compared to controls (t = 3.3, t = 3.0, t = 2.0 for
left anterior hippocampus and body of the hippocampus and the left anterior thalamus respectively; all surviving 5% FDR), although the absolute value of these volumetric increases do not translate to increased
total hippocampal volume. Local volume changes showed higher variability in the sham group in comparison to the DBS group (Bartlett's
K2 = 2.93, p = 0.086 for hippocampus).

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425

Fig. 1. A) Mice receiving DBS show larger i) bilateral hippocampus volume and ii) left thalamic volume. No volumetric differences were observed between sham and control groups at the whole
structure level. B) These ﬁndings of overall volume increases can be localized to local volumetric expansions in both the i) hippocampus (bilaterally) and the ii) left thalamus. Note that open
circles in B) represent individual data points that do not fall within the bounds of the box and whisker plots. Signiﬁcant results that are observed in the superior margin of the brain are due toground screws implanted for the DBS stimulator. # and ## denote survival of 10% and 5% false discovery rate correction.

Subsequent histological analysis in these regions demonstrated
evidence of neuroanatomical remodeling. The DBS group showed
increased hippocampal blood vessel diameter relative to controls(collagen, t = 4.21; p b 0.001; see Fig. 2A, ii). Synaptic density(synaptophysin) was increased relative to controls in both the DBS(t = 3.18; p b 0.001) and sham groups (t = 2.86; p b 0.005; see
Fig. 2A, iv). In addition, there was evidence of increased astrocytearea in the hippocampus in the sham group compared to controls

Fig. 2. A) Subsequent evaluation using histological preparations demonstrate that mice receiving DBS i) sample slide demonstrating a triple stained image using collagen (green), GFAP (red),and DAPI (blue) ii) have larger blood vessels (collagen stain), while the iii) sham group demonstrates larger astrocytes (GFAP stain). Mice receiving DBS also show iv) increased synaptic density(as measured using synaptophysin). B) Signiﬁcant correlation of the i) mean vessel area and the ii) mean astrocyte area with the relative change in voxel volume was observed at the peaklocation of volumetric expansion in the hippocampus. Note that open circles in A) represent individual data points that do not fall within the bounds of the box and whisker plots. *, **, ***are statistically signiﬁcant at p b 0.05, b0.005, and b0.001.

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M.M. Chakravarty et al. / NeuroImage 125 (2016) 422–427(t = 3.16; p b 0.005; as measured using GFAP; see Fig. 2A, iii). Blood vessel (t = 3.19, p b 0.05, R2 = 0.28) and astrocyte (t = 2.24, p b 0.05, R2 =0.25) size were well correlated with hippocampal volumetric expansion
observed on MRI and accounted for the largest proportion of the overallvariance in volume (see Fig. 2B).
Discussion
In this manuscript we demonstrate evidence that both the implantation of a DBS electrode and active simulation via DBS in the rodent
homologue of the subcallosal cingulate region is associated with neuroanatomical remodeling at the level of the density of synaptic connections.
We further demonstrate that active DBS may be involved in remodelingof the vascular architecture, resulting in increased blood vessel diameter.
Traditionally, DBS has been considered a purely symptomatic therapy, unable to inﬂuence brain structure. However, these results, may be seen ascomplementary to other results from our group (Hamani et al., 2011;
Stone et al., 2011). They demonstrate that both the insertion of the DBS
electrode and active electrical stimulation may induce distinct and overlapping neuroanatomical remodeling at the cellular level. Importantly,
these results also demonstrate that the neuroanatomical rearrangement
in response to DBS can occur distal from (i.e., either upstream or downstream of) the stimulation site. In other words, DBS impacts components
of neuroanatomical networks that are connected to the stimulation sitethrough multiple synapses (Stone et al., 2011). Our observation of neuroanatomical rearrangement in both the hippocampus and the anterior
thalamus in response to DBS of the VMPFC provides further support forthis notion. In fact, further exploration using the Allen Institute Mouse
Brain Connectivity Atlas (Oh et al., 2014) demonstrates connectivity between the VMPFC, the anterior portions of the thalamus, and hippocampal regions such as CA1 and CA3 (regions where volumetric expansion
was found in our study using volumetric MRI; see Supplementary
Figure S2).
Our results also suggest that neuroplasticity may be vital to the efﬁcacy of DBS for depression and further suggest that the hippocampal growthobserved may be necessary for providing an anti-depressant effect. Whilehippocampal decreases have previously been reported in TRD (McKinnon
et al., 2009), there have been reports of 5% bilateral hippocampal growth
in refractory patients as an acute response to electroconvulsive therapy(Nordanskog et al., 2010). Studies of depressed non-human primateshave observed decreased neuropil and cell layer volumes in the CA1
and dentate gyrus regions of the hippocampus (Willard et al., 2013). Interestingly rat depression models treated with another form of brain
stimulation, electroconvulsive seizure treatment, demonstrate an increased number of glial cells in the hilus of the hippocampus (Kaaeet al., 2012) and an increase in the total number of synapses (Chenet al., 2009); this remodeling was also associated with antidepressantlike effects in these rats. Other preclinical studies also note speciﬁc alteration and increases in hippocampal volumes after the administration ofanti-depressant medications (Venna et al., 2009); while some pharmacological treatments (such as imipramine) may modulate the establishmentof new synaptic connections and may actually remodel existing synapses
by increasing modulatory spine synapses and altering the normative distribution of spine to shaft synapsis (Chen et al., 2008).
Our results also demonstrate that neuroanatomical remodeling occurs
even in response to a sham DBS insertion, suggesting that improvements
seen clinically after DBS insertion (Cersosimo et al., 2009; Granziera et al.,2008) may not entirely be caused by a placebo effect. However, whileboth active and sham DBS cause increased synaptic density, there appears
to be potential reactive gliosis (much like the effects previously reported
in response to a needle insertion to the hippocampus (Song et al., 2013))
in response to the sham stimulator. It is possible that these mechanisms
are being further modulated through active stimulation. While purely
speculative, it is also possible that the sham group is experiencing the increase of neutrophic factors often seen in the mammalian brain in response to acute injury or lesions (Needels et al., 1985, 1986).

Our ﬁnding of increased collagen staining following VMPFC DBS
leads us to speculate that the phenomenon of increased cerebral blood
ﬂow observed at 6 months in TRD patients receiving subcallosal DBS
may be mediated by remodeling at the vascular level (Lozano et al.,2008). Interestingly, our ﬁndings and the ﬁndings from the initial clinical trials are at odds with measurements of blood ﬂow in response toother forms of brain stimulation such as transcranial direct current
stimulation in humans (Stagg et al., 2013) that demonstrate decreases
in cortical perfusion as a result of both anodal and cathodal stimulationsof the dorsolateral prefrontal cortex. Similarly, DBS of the rat hippocampus decreased hippocampal regional cerebral blood ﬂow as evaluatedby single photon emission computed tomography (Wyckhuys et al.,2010). Conversely, there is evidence of artery dilation as a result of
focal electrical stimulation in the nucleus basalis of Meynert based on
two-photon microscopy experiments; however this stimulation occurred at a lower frequency (50 Hz) (Hotta et al., 2013). Despite these
conﬂicting previous results, there is little in the way of investigation between the coupling between DBS and alterations in vascular morphometry and arborization and alteration in the dynamics of cerebral blood
ﬂow. The differential effects described above suggest that any changes
in cerebral blood ﬂow mediated by brain stimulation may be dependent
on the type of stimulation, the frequency of the stimulation, and thestimulation target. Clearly, further investigation along these lines is required to better understand the coupling between blood ﬂow, vascularalterations, and the application of DBS.
Finally, there are limitations to consider in the interpretation of theresults that are presented in this manuscript. Our work only observes
differences between stimulated, sham treated, and control animals in
response to stimulation for a limited number of hours each day. Froma translational neuroscience perspective, it would have been beneﬁcial
to closely mimic the clinical DBS protocol where chronic stimulation isadministered 24 h/day (Lozano et al., 2008; Mayberg et al., 2005;
Kennedy et al., 2011). Unfortunately mimicking this scenario was not
feasible, and we thus opted to stimulate the mice for a limited numberof hours; a paradigm that has shown to induce anti-depressant effects
in rats in previous work from our group (Hamani et al., 2010). To thatend it is unclear the extent to which neuroanatomical plasticity may
be linked to the anti-depressant-like effects previously reported clinically. Another way to test the association between neuroanatomical
remodeling would have been to administer a forced swim test as previously done by our group (Hamani et al., 2010, 2012) in order to provide
putative associations between the observed result and behaviouralchanges. As a result associations between our results (and those ofmany preclinical studies) and their possible translation to clinical practice should be considered speculative. However, given the literature
reviewed above it is plausible that there is an association between neuroanatomical remodeling observed and therapeutic effects previously
observed.
In summary, ours is the ﬁrst study to combine MRI and histology
in the context of experimental DBS. We demonstrate that volumetricchanges that can be resolved using MRI do indeed accompany stimulation, and are further correlated with speciﬁc neuroanatomical
processes, especially those related to blood ﬂow and neuronal synaptic connectivity. Our ﬁndings suggest the exciting possibility ofcompensatory or even neuroregenerative mechanisms initiated byelectrical stimulation, and the possible role of the micro-lesion inacute post-operative response in depressed patients prior to the initiation of active stimulation. Further work is needed to elucidate theextent, time course, and patterning of neuroanatomical changes accompanying DBS for depression and other illnesses.
Acknowledgments
M.M.C. is supported by funding from the Weston Brain Institute, Michael J. Fox Foundation, Alzheimer's Society, National Sciences and Engineering Research Council of Canada, Canadian Institutes of HealthM.M. Chakravarty et al. / NeuroImage 125 (2016) 422–427

Research, and Brain Canada. M.M.C receives salary support from theFonds de recherches Santé Québec (Junior 1 Scholar Program). T.S. is supported by a Canadian Institutes of Health Research (CIHR) fellowshipaward. J.P.L is funded by the CIHR and Brain Canada.
Disclosures
M.M.C: none, C.H.: none, A. M-C.: none, J.E.: none, C.L.: none, J.N.N.:
none, T.S.: none, A.M.L.: is a consultant to Medtronic, St. Jude, and Boston
Scientiﬁc. A.M.L. serves on the scientiﬁc advisory board of Ceregene,
Codman, Neurophage, Aleva and Alcyone Life Sciences. A.M.L. is a cofounder of Functional Neuromodulation Inc. and holds intellectual property in the ﬁeld of Deep Brain Stimulation. P.W.F.: none, J.P.L.: none.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.neuroimage.2015.10.049.
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