Investigation of Cortico-limbic Networks and Their Dynamics in Major Depressive Disorder
Phase: NA
Conditions: Major Depressive Disorder, Recurrent
Sponsor: Massachusetts General Hospital

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Title: Investigation of Cortico-limbic Networks and Their Dynamics in Major Depressive 
Disorder  
 
Principal Investigator: Mark Eldaief, MD 
Co-Investigators: Randy Buckner, PhD; Jacob Hooker, PhD, Darin Dougherty, MD, Michael 
Fox, MD, PhD, Ciprian Catana, MD, PhD, Tommi Raij, MD, PhD 
Other Study Staff: Shirley Hsu, Grae Arabasz, Regan Butterfield 
Non-Partners collaborator: Alvaro Pascual-Leone, MD, PhD  
 
I. 
BACKGROUND AND SIGNIFICANCE 
The neurobiology of Major Depressive Disorder (MDD) remains unclear. Functional neuroimaging approaches 
such as Positron Emission Tomography (PET) and functional MRI (fMRI) have delineated abnormalities in 
resting metabolism and task-based activation in this condition.1-3,23,25,26 Affected cortico-limbic regions include 
those involved in: emotional processing [e.g., amydala and nucleus accumbens (NA)], visceromotor control 
and homeostasis (e.g., insula and hypothalamus) and emotional regulation [e.g., orbitofrontal (OFC) and 
dorsomedial (dmPFC) prefrontal cortex]. Consistent changes are also seen in the subgenual cingulate 
(sgACC, BA25) and in the dorsolateral prefrontal cortex (DLPFC), wherein PET studies have revealed 
hypermetabolism and hypometabolism1-4, respectively.    
The observation that dysfunctions are present in distributed cortico-limbic regions suggests that MDD 
symptoms might evolve from aberrations in distributed processing between the regions in question, in other 
words, at the level of neural networks. Building on this possibility, the use of network measures may provide 
valuable endophenotypes of psychiatric diseases such as MDD.13 Consequently, there has been increasing 
interest in characterizing the functional architecture of neural networks in depression1-7, especially those 
encompassing prefrontal and subcortical regions heavily implicated in its pathogenesis. 
Functional connectivity MRI (fcMRI) relies upon correlations between spontaneous low frequency fluctuations 
in the blood oxygenation level-dependent (BOLD) signal to delineate intrinsic brain networks in the absence of 
explicit task demands.27-29 fcMRI studies in normals have revealed topographically consistent patterns of 
functional connectivity among the aforementioned cortico-limbic regions. Using seed based analytic 
approaches, sgACC  has been shown to be functionally correlated to several limbic regions including the OFC, 
amygdala and NA .9,21,22,30 This functional connectivity strongly recapitulates structural projections to and from 
the sgACC in animal and human studies.31-33 In addition, sgACC shares functional connectivity with portions of 
the default network (DN), specifically the dmPFC9, the temporal pole and the posterior cingulate/precuneus 
(pCC).21,22 Relevant to this study, sgACC is functionally correlated to a medial portion of the DLPFC (superior 
frontal gyrus, a part of the DN), but is anticorrelated to another portion of DLPFC (middle frontal gyrus).21,22 
These correlation patterns have been borne out within our own data (Fig 1). 
 
 
 
Fig 1. Voxel-wise functional connectivity maps derived from seeding 
the sgACC in 26 healthy subjects in our lab. In DLPFC, both positive 
(red/yellow) and negative (blue) correlations are observed.  
In MDD, evidence of aberrant network activity was 
first described in the form of reduced deactivations in 
the DN during task execution.34-35 With regards to 
functional connectivity, studies suggest that the 
sgACC and dmPFC9 are excessively coupled to their 
distributed cortico-limbic partners. Resting-state fMRI 
data have also elaborated abnormal network metrics 
in MDD, including increased regional homogeneity, 
increased nodal centrality and connectivity degree, 
and decreased small-worldness.36,37 Notably, these 
metrics were identified in similar cortico-limbic and 
default regions, and they correlated with disease 
severity. 

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Greicius and colleagues8 employed Independent Component Analysis (ICA) to demonstrate that DN functional 
connectivity was significantly increased in the sgACC, medial thalamus and OFC in MDD patients, and that 
increased sgACC connectivity correlated with duration of depressive episodes. Two other fcMRI studies (one 
using seed-based fcMRI, and another using ICA) correlated rumination scores in MDD patients to increased 
sgACC functional connectivity.10,11 In addition, Sheline et al. used seed-based approaches to demonstrate 
heightened connectivity between sgACC and dmPFC in MDD patients.9 
However, despite efforts to capture local abnormalities with PET, and network abnormalities with fcMRI, we 
currently lack integrative models placing these cortico-limbic nodes into dynamic systems (i.e., as they are 
likely to exist in the in vivo brain). The notion of “network dynamics” is supported by novel computational 
modeling approaches14,38 which have uncovered temporal changes in functional connectivity over the course of 
fcMRI scanning sessions.39-40 Taken in this light, complex interplays between local nodal processing and 
functional couplings between network nodes are constantly at work. In this way, connectivity between network 
nodes is conceived as being in a “dynamic equilibrium,” in which small conformational departures occur rapidly 
and continuously, but in which steady states of network connectivity are realized over long time scales.12,13  In 
MDD, the threshold for depressive symptoms might be lowered when this network equilibrium is shifted-
causing abnormally high processing through limbic regions. Alternatively, mechanisms which normally 
compensate for these shifts might be aberrant in MDD. Within this framework, one might expect that 
interventions which restore such dynamic inequities might offer therapeutic promise. 
One such intervention is repetitive transcranial magnetic stimulation (rTMS). rTMS delivered to DLPFC is an 
established treatment for MDD, with its clinical efficacy supported by large randomized, sham controlled 
trials15,16, and several meta-analyses.41-43 Also, the Neurostar rTMS treatment (Neuronetics®) has been FDA 
approved for certain forms of medication-refractory depression. Broadly, the effects of rTMS are known to 
propagate, via trans-synaptic means, to distal but interconnected regions with spatial specificity.44,45 This 
suggests that it exerts its anti-depressant effects through re-modeling of cortico-limbic networks. That rTMS to 
DLPFC impacts medial limbic structures is also supported by H215O PET studies in normals showing that rTMS 
to DLPFC changes regional cerebral blood flow (rCBF) in ipsilateral anterior cingulate cortex and in the 
sgACC.46,47 Also, another protocol used PET to demonstrate that DLPFC stimulation modulates 
neurotransmitter function in these regions.48 Nonetheless, in spite of its efficacy in MDD, and despite evidence 
that its effects are mediated through trans-synaptic influences on limbic structures, the effects of rTMS to 
DLPFC upon cortico-limbic functional connectivity have yet to be characterized. 
                                                                                                            
 
 
 
 
Preliminary data: My prior work reveals that rTMS combined 
with fcMRI offers the ability to probe the dynamics of cortico-
limbic networks. In Eldaief et al.14 we studied healthy young 
participants who underwent neuronavigated rTMS to the left 
posterior inferior parietal lobule (lpIPL) node of their DN. First, 
subjects had a baseline resting-state fcMRI scan to individually 
identify an lpIPL target for rTMS, based upon their own fcMRI 
data. After this baseline, subjects returned for two additional 
sessions during which they had resting state fcMRIs before, 
and after, rTMS to lpIPL. In one session rTMS was delivered 
with low frequency (1 Hz) stimulation, and in another with high 
frequency (20 Hz) stimulation. Stimulation parameters (e.g. 
number of pulses and stimulation intensity) were identical 
across sessions and stimulation durations were comparable. 
We used a stereotactic rTMS neuronavigation system to 
ensure that the same lpIPL target was reliably stimulated 
during both sessions. After 20Hz rTMS to lpIPL, functional 
correlations decreased between lpIPL and other cortical 
regions of the DN (e.g. medial prefrontal cortex, mPFC, and 
pCC). 20 Hz rTMS did not alter functional connectivity between 
these regions and the hippocampus (HF). However, 1Hz rTMS 
increased functional connectivity between pIPL and HF, but not 
between lpIPL and other cortical DN nodes (Fig 2). This study 
demonstrates that different rTMS regimens to the same DN 
node elaborated two different functional network configurations.  
Fig 2.  Functional connectivity maps derived from an 
lpIPL seed location at baseline (top). Voxelwise paired 
t-test maps representing changes in functional 
connectivity correlations are also shown after low 
(middle) and high (bottom) frequency rTMS. 
Connectivity decreases are shown in blue and 
increases in red/yellow.   

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More broadly, this study established the safety and feasibility of modulating intrinsic network activity with non-
invasive brain stimulation. Dr. Pascual-Leone’s (advisor) group has corroborated this by recently showing that 
active transcranial direct current stimulation (tDCS) to DLPFC decreases DN synchrony, but increases 
connectivity in an anticorrelated network.49 Other research groups have published consistent results50: Keeser 
et al. showed that anodal stimulation of DLPFC with tDCS modulated connectivity in the DN51; Alon et al. found 
that excitatory stimulation of M1 with tDCS decreased intrinsic connectivity between bilateral M1.52  
Nevertheless, further exploration of intrinsic network dynamics using rTMS and fcMRI would benefit from a 
greater understanding of the physiologic effects of rTMS upon the directly targeted brain region. In our 
preliminary study, connectivity changes may have been predominantly determined by stimulation dependent 
changes in excitability at lpIPL. While prior studies suggest that high and low frequency rTMS exert opposing 
effects on cortical excitability53-55, we could not verify the magnitude, directionality or diffusivity of the 
stimulation at lpIPL. Another consideration is that tonic activity at lpIPL or at distal network nodes, might 
constrain the extent to which connectivity is modulated with rTMS.56 This is especially germane to MDD, given 
the presence of baseline metabolic derangements at DLPFC and sgACC. An ideal way to circumvent this 
shortcoming is to simultaneously accrue information about changes in local nodal activity and changes in 
network connectivity. 
 
This study will take advantage of a novel combined MRI-PET scanner19,20 (the MRI-BrainPET camera) to 
simultaneously record BOLD fcMRI and 18Flurodeoxyglucose (FDG)-PET data following rTMS to DLPFC, in 
both depressed patients and in healthy controls. We will specifically target regions of the DLPFC which share 
functional associations with the sgACC. The sgACC was chosen as the main limbic region of interest because 
of mounting evidence for the pivotal and integrative role it plays in MDD. For instance, sgACC activity is 
enhanced during acute sadness57 and reduced after anti-depressant regimens.58,59 Also, the sgACC is the 
target of deep brain stimulation (DBS) in treatment refractory MDD.33,60 Network dynamics in MDD will be 
assessed by comparing identical stimulation regimens delivered to regions correlated, and anticorrelated, with 
sgACC, (analogous to how we previously used two different rTMS regimens to lpIPL14). Motivation for inclusion 
of the correlated region is self-evident based on our prior study.14 Motivation for stimulating the anticorrelated 
region is twofold. First, sgACC anticorrelations are rather prominent in DLPFC.21,22 Second, these tend to be 
more lateral, and thus anatomically more closely approximate the clinical stimulation target.17,61,62 Primary 
outcomes of interest will be rTMS induced changes in regional cerebral metabolic rate of glucose uptake 
(rCMRglu) at the DLPFC stimulation sites and in sgACC; as well as rTMS induced changes in functional 
connectivity between sgACC-DLPFC. Secondary outcomes will involve rTMS induced connectivity changes 
between sgACC and three additional cortico-limbic nodes: dmPFC9, NA1-5 and amygdala.30 Network dynamics 
will be assessed through observation of the interplay between local metabolic and network changes. While 
extant data63 might be predictive of these dynamics (Fig 3), we expect that these will be revealed empirically. 
  
 
 
 
 
 
 
 
 
 
 
Fig 3. Schematic representation of how local and 
distributed changes induced by rTMS to DLPFC 
might interact. (A) No rTMS, (B) 20Hz rTMS to 
DLPFC positively correlated to sgACC and (C) 
20Hz rTMS to DLPFC anticorrelated to sgACC. 
In this model, 20Hz rTMS increases rCMRglu at 
both DLPFC sites (up arrows)53-55. In B, this 
might decrease connectivity between DLPFC and 
sgACC (thinned red line) and increase rCMRglu 
in sgACC. In C, rTMS decreases rCMRglu in 
sgACC and increases anticorrelation between 
these regions (thickened blue line). 

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II. SPECIFIC AIMS 
Aim 1: To assess rTMS induced changes in functional connectivity in cortico-limbic networks 
Hypothesis 1.1: At baseline, MDD patients will have abnormally increased cortico-limbic connectivity.8-11 
Hypothesis 1.2: rTMS to DLPFC will differentially modulate this connectivity, depending on whether positively 
or negatively correlated regions are targeted; and one of these targets will more robustly alter this connectivity. 
Hypothesis 1.3: Connectivity changes will be more pronounced in MDD patients than in controls. 
Aim 2: To assess cortico-limbic network dynamics in MDD by comparing rTMS induced changes in 
local glucose metabolism to rTMS induced changes in distributed connectivity  
Hypothesis 2.1: 20Hz will comparably increase FDG uptake at both DLPFC stimulation sites in both groups. 
Hypothesis 2.2: Stimulation of the two sites will differentially impact FDG uptake at sgACC, and will have a 
differential impact upon the strength of connectivity between sgACC and the stimulation site. 
Hypothesis 2.3: The direction and magnitude of FDG changes at sgACC will predict the direction and 
magnitude of the associated changes in cortico-limbic connectivity. 
Hypothesis 2.4: Local and distributed changes will be altered in MDD, reflecting aberrant network dynamics. 
Hypothesis 2.5: Stimulation of a control site in parietal cortex or the cerebellum will not appreciably alter cortico-
limbic connectivity or metabolism. 
This work will better elucidate how intrinsic networks and their dynamics are altered in MDD. It will also assess 
how rTMS, or other neuromodulatory strategies, create the formation of more adaptive configurations. In turn, 
this will pollinate future trials that optimize MDD treatments through selective targeting of intrinsic networks. 
 
 
III. SUBJECT SELECTION 
 
A. MDD patients: 25 patients (ages 18-50) with unipolar MDD (single episode or recurrent) will 
participate. We will specifically recruit unmedicated patients. At enrollment, patients must be medication 
free for a period consistent with four half-lives of the drug in question (e.g. 4 weeks for fluoxetine). 
Diagnoses of MDD will be made by each patient’s referring physician, or, in the case that there is not a 
referring physician, will be made by the PI (a board certified psychiatrist) with DSM-IVR criteria using 
the Structured Clinical Interview (SCID).65 Patients will also be required to score ≥187 on the 24 item 
version of the Hamilton Depression Rating Scale (HDRS).24 All psychometric assessments will be 
performed by a trained technician. Patients with any of the following issues will be excluded: bipolar 
disorder, schizoaffective disorder, active suicidal ideation, or any history of psychosis. Given the high 
co-morbidity, concurrent anxiety disorders will be allowed, provided MDD is the major diagnostic 
consideration.    
B. Healthy subjects: 25 healthy controls (ages 18-50) will also be recruited. These subjects will be 
matched to MDD patients on the basis of age, gender and ethnicity. Controls must score ≤8 on the 
HDRS. These subjects must not meet criteria for any Axis I disorder (confirmed by DSM-IV criteria and 
by the SCID questionnaire), have no formal history of these, and must not be on any psychotropic 
medications.  
C. Exclusion Criteria (All Subjects) 
1. A history of serious concurrent medical or neurological illness, especially any patient with a known 
space-occupying lesion in the brain/skull. 
2. Concurrent substance use, abuse or dependence.  
3. Contraindications to receive MRI scanning including, but not limited to the presence of metallic or 
ferromagnetic implants (e.g. pacemaker, defibrillator, vagal nerve stimulator, implantable pump, 
metallic tattoos and/or make-up, surgical aneurysm clips, etc.); or severe claustrophobia. 
4. Contraindications to undergo PET scanning, e.g. a history of receiving radiation exposure with PET 
within the past 12 months.  
5. Contraindications to receiving TMS, specifically a history of seizures/epilepsy in the subject or in a 
first-degree relative or history of unexplained syncope. 
6. Any subject who is pregnant (as assessed by a STAT quantitative serum hCG test) or currently 
lactating. 

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7. Any subject who has known metallic or ferromagnetic bioimplants, or who has a history of seizures 
or a first degree relative with seizures, or who has a history of significant claustrophobia. 
 
D. Sample Size Determination: Sample size calculations for the rTMS/fMRI studies are based on Eldaief 
et al, as we are unaware of other studies that provide empirical estimates of effect size. The main 
outcome measure for the TMS/fMRI experiments will be a change (pre vs. post-rTMS) in correlation 
values (z) between sgACC and DLPFC. Our study of 17 subjects yielded an effect size of 0.15 for 20Hz 
rTMS induced changes between lpIPL and mPFC (SD=0.11). Based on this, sample sizes of 8 and 12 
would have enough power (90%) to detect changes similar to those observed in our prior study at a 
significance level of 5% using an unpaired t-test or a paired t-test, respectively. With respect to PET, a 
comparable FDG-PET study66 found an average change in rCMRglu of 5.3% (SD=2.77%) following 
high frequency rTMS. Based on this, sample sizes of 5 and 7 would be required (power, 90%; α, 0.05; 
unpaired and paired t-tests, respectively). However, because these effect sizes were all calculated from 
healthy controls, they might represent underestimates of the effect sizes needed for MDD patients. 
Therefore, we will conservatively recruit 25 subjects for each subject group.  
A. Source of Subjects/Recruitment Methods: Healthy participants will be recruited via bulletin board 
announcements (please see advertising materials) placed throughout the Brigham and Women’s 
hospital, Massachusetts General Hospital, MGH/Martinos Center, Harvard University Countway Library 
and Harvard School of Public Health, which will be first approved through the PHRC. There will also be 
two online advertisements, one through the website Craigslist and another through the Boston 
University job site “Quickie Jobs” (www.bu.edu/seo/studentjobservice/quickie-jobs).  MDD patients will 
be recruited through all of these sources via a separate advertisement (please see advertisement 
materials). We will target individuals who are not currently undergoing treatment for their current major 
depressive episode. Prospective depressed subjects will be given a contact number and email in order 
to allow them to seek out more information about the study. They will then undergo a brief PHRC 
approved telephone survey/email survey (see attachments) with the PI to establish their eligibility. If 
they are considered eligible, they will be invited to participate in the first study visit where they will be 
consented in written form prior to undergoing study procedures. Other recruitment sources will for MDD 
patients will include individuals presenting to Partners sites for evaluation and management of 
depressive symptoms. Specifically, this will be comprised of three sources: 
1. The BWH outpatient psychiatry department. In this case recruitment will be accomplished through a 
separate recruitment letter to subjects’ primary psychiatric providers (please see separate 
attachment). The recruitment letter will be cosigned by the patient’s primary psychiatrist and by the 
PI. The letter will include an "opt out" in which subjects will be permitted to call to opt out of study 
participation and not be contacted.  
2. Patients of Dr. Eldaief (PI) who present to the Division of Cognitive and Behavioral Neurology with 
symptoms of major depression. In order to minimize coercion of potential subjects who are patients 
of the PI, patients will be offered a copy of the Consent Form and be instructed to call back if they 
are interested in participating. In addition, the PI will strongly recommend that they discuss their 
potential study participation with other health care providers (e.g. their primary care physician and 
psychiatric provider). 
3. Patients of Dr. Dougherty (co-investigator) who present to the MGH Depression Clinical Research 
Program (DCRP) with symptoms of major depression.  
 
In the latter two cases, the PI will strongly emphasize that study participation is voluntary and that 
subjects’ decisions to not participate will in no way affect their care with said investigator or their future 
care at any Partners institution.  
 
Once identified as potentially eligible, subjects will undergo the aforementioned telephone screening/email 
assessments with the PI to further delineate their eligibility. 
 
 
 

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IV. SUBJECT ENROLLMENT 
 
A. Subjects will be randomized as to the order of the TMS sessions they undergo (there are two types of 
sessions in which one of two sites on the prefrontal cortex is stimulated). The order of these sessions 
will be counterbalanced across subjects. In other words, we will alternate between the two types of 
sessions and will have equal numbers of subjects having undergone a particular order of sessions. This 
will allow us to account for order effects.  
Informed consent will be obtained by the principal investigator during the screening visit. Participants will 
be told that this is an MRI/PET study combined with TMS stimulation. The interviewer will describe the 
MR/PET procedures in depth, the TMS instruments in depth, and any risks previously described. These 
descriptions will include: how long the participant is expected to remain inside the magnet, how long the 
TMS sessions will be, and minor risks associated with exposure to radiation from the FDG ligand. Subjects 
will also be informed about the small space within the magnet and noises made by switching gradients.  
Subjects will also be told that the MRI procedure is NOT diagnostic, and that any abnormalities detected 
will be referred to a consulting radiologist for review.  They will be told that the radiation they receive from 
PET scanning is not necessary for their medical care and is for research purposes only. In addition, the 
participant will be told how many experimental visits he/she is expected to complete and the duration and 
timing of these visits will be explicitly conveyed. Subjects will read the consent form without any time 
constraints. The subject will also be offered to take the consent form home and think about participation. 
Any and all questions will be answered to the best of the investigator's abilities. Every effort will be made to 
come up with clear, factual answers to all questions. The subject will be asked to summarize the procedure 
and recount it to the interviewer as a robust assessment of the subject's understanding of it.  
Because there are not well validated versions of the questionnaires employed (e.g. the SCID and HDRS 
scales) in other languages, subjects who do not speak English will be excluded from study participation.  
The subject will also be informed that, at any point, he/she may choose to terminate the study for any 
reason and that he/she has the option not to participate in the study. This includes being able to terminate 
the study during the experimental procedures proper. They will be informed that their refusal to participate 
in the study, or their wish to terminate it at some point, will have no effect on care and treatment received 
by them at any Partners hospital including the Brigham and Women’s hospital and the Massachusetts 
General Hospital or any of their affiliated institutions, now, or at any time in the future. Should the subject 
indicate that he/she wishes to participate in the study, written informed consent will be obtained. All 
participants will be provided with copies of the signed consent form. 
 
V. STUDY PROCEDURES 
 
A. rTMS, PET and fMRI procedures: 
fcMRI and PET will be acquired using an integrated Siemens 3.0 
T whole-body Siemens MRI scanner fitted with a 3D dedicated 
BrainPET camera housed at the Athinoula A. Martinos Center for 
Biomedical Imaging at the MGH Charlestown Navy Yard. This 
scanner is capable of the simultaneous acquisition of PET and 
MRI data (Fig 4). 
 
fMRI procedures: Structural images will be acquired via a 3D-
turbo field echo sequence which will generate high resolution 3D 
MPRAGE T1-weighted images. Functional images will be 
acquired using a T2*-weighted sequence. Functional data will be 
collected by using an asymmetric spin-echo, echo-planar 
sequence sensitive to BOLD contrast during three 6 minute 
functional runs while subjects rest and stare at a white fixation 
MRI 
Fused MR-PET 
PET 
Fig 4: Simultaneous MR-PET data acquisition in a 
healthy subject using the MRI-BrainPET scanner. 
Axial, coronal and sagittal FDG-PET (upper), fused 
MR-PET (middle) and MR (lower) are shown. 

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dot. During this time, subjects will be instructed to lay as still as possible and “think about whatever they want.” 
 
PET procedures:  
Subjects will be instructed to fast and not to consume beverages, except for water, for at least 4 hours before 
the administration of FDG to decrease physiologic glucose levels and to reduce serum insulin levels near basal 
levels.  While fasting, subjects should consume at least two to three 8-12oz glasses of water to ensure 
adequate hydration.   
 
Subjects will also be instructed to avoid strenuous exercise for 24 hours before the FDG-PET to minimize 
uptake of the radiotracer in the muscles. A fingerstick will assess the blood glucose level before FDG 
administration to ensure it is below the specified limit (i.e. 200 mg/dl) 
 
An intravenous catheter will be placed and will be flushed post-injection of FDG with 0.9% saline solution. 
Participants will receive an intravenous bolus of 5mCi (~185 MBq) of 18Flurodeoxyglucose (FDG) by a trained 
technician (specifically Shirley Hsu or Grae Arabasz, both study staff). Injection will occur coincidentally to the 
onset of rTMS. Dynamic scanning will begin approximately 30 minutes after this so that dynamic changes in 
rCMRglu can be modeled67 during the anticipated peak effects of rTMS.  Emission data will be acquired over a 
45 minute period and stored in list-mode format. Data will first be sorted in the line-of-response (LOR) space 
and motion correction will be applied using MRI-derived motion estimates for each individual frame. Next, data 
will be rebinned in the sinogram space, generating prompt and random coincidences sinograms. 
 
rTMS procedures: rTMS will be administered with a Magstim Rapid System, using a 70 mm figure-of-eight air-
cooled coil. For allsessions, 20Hz stimulation will be delivered at 110% of resting motor threshold via 75 trains 
of 1.2 second 20Hz  rTMS (i.e., 24 pulses per train), each of which will be followed by inter-train pauses of 16.8 
seconds for a total of 1800 pulses (22.5 min), for a total of 1800 pulses (22.5 min). These parameters are 
within recommended safety limits for rTMS68, and replicate parameters used in Eldaief et al.14 Similar rTMS 
durations have been shown in other paradigms to outlast the stimulation period by anywhere from 20 
minutes53,54 to up to 1 hour.69 During rTMS, accurate targeting of DLPFC, or the control site, will be achieved 
with a frameless stereotactic optical tracking neuronavigation system (Brainsight™), which will permit real-time 
monitoring of coil placement and head position.70 
 
B. Eligibility screening and baseline questionnaires:  
Study procedures will first consist of completion of study questionnaires followed by experimental 
procedures. In order to ensure eligibility to participate, all subjects will complete a SCID and HDRS rating 
scale. These will confirm the existence/absence of major depression and the absence of comorbid 
psychiatric conditions. Other exclusionary criteria will be screened via non-structured clinical interviews. A 
subject’s eligibility for safely receiving rTMS, PET and fMRI will be assessed by filling out the appropriate 
screening forms (see attachments). All of these procedures will be performed by Dr. Eldaief (PI) who is 
trained in administering these instruments and screening forms.  Finally, women of childbearing age will 
undergo a STAT quantitative serum hcG to ensure that they are not pregnant. 
C. Aim 1 and 2 Experimental Procedures 
                             
50 subjects (25 MDD patients and 25 matched controls) will each undergo three experimental visits. In addition, 
of these subjects a subset (no more than five) will be given the option to undergo a fourth experimental visit. 
During the first study visit, consent will be obtained. Following this, subjects will be assessed for eligibility by 
Fig 5. 
Experimental 
timeline. Baseline 
fMRI/PET done 
on visit 1. 
Subjects return 
days later for 2-3 
more visits in 
which they have 
fMRIs before 
rTMS and 
fMRI/PET 
afterwards. 

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screening procedures mentioned above. If they are considered eligible, they will undergo a baseline scan during 
this visit. 
 
Specifically, subjects will undergo simultaneous resting-state (fcMRI)/FDG-PET imaging without rTMS at this first 
visit. The baseline PET session will serve as a comparison for changes in rCMRglu following rTMS. Critically, 
strong evidence supports the test re-test reliability across FDG-PET scanning sessions separated in time, 
provided that scanning parameters remain identical.71-74 Therefore, observed metabolic changes after rTMS will 
be attributable to the stimulation, and will be deemed unlikely to occur as a result of inherent variability across 
scanning sessions. Baseline fcMRI data will serve the purpose of creating functional connectivity maps from a 
sgACC seed. Similar to how lpIPL was designated in Eldaief et al.14, these maps will be used to establish two 
cortical targets for rTMS in DLPFC: one which is positively correlated to sgACC, and another which is 
anticorrelated to sgACC21,22 (Fig 1). After their baseline visit, subjects will return for the first of two rTMS 
sessions in which they will receive 20Hz stimulation to either the positively or negatively correlated left DLPFC 
region. They will then return for a third session in which the alternate region is stimulated (thus enabling a within-
subject design). Subjects will be randomized to a given session order, and this order will be counterbalanced 
across subjects. It is expected that all three visits will be completed within two months from the time of 
enrollment. The experimental timeline for the rTMS sessions is depicted in Fig 5. Immediately before stimulation 
subjects will be rapidly scanned with resting-state fMRI without PET (for approx. 25 minutes). After this, they will 
fill out a brief (1 min) visual analog scale in which they rate sadness, anxiety, energy, concentration, happiness, 
anger and disgust on a scale of 0-10. They will then be taken into a room immediately adjacent to the scanner 
for rTMS (i.e. constituting “off-line” stimulation75). Subjects will be injected with FDG concurrent to the onset of 
rTMS. After rTMS they will again complete the mood analog scale. They will then be promptly returned to the 
scanner room for PET/fcMRI (within ~5 min) so as to acquire BOLD data as soon as possible after rTMS.14 
Dynamic PET acquisitions will correspondingly begin at this time (e.g. at approx. 30-35 min post FDG injection), 
and emission data will be collected for the next 45 min. 
 
 
 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Obtainment of Consent 
X 
 
 
 
SCID questionnaire 
X 
 
 
 
Hamilton Depression Rating Scale (HDRS) 
X 
 
 
 
TMS screening form 
X 
 
 
 
MRI screening form 
X 
X 
X 
X 
STAT quantitative serum hcG 
X 
X** 
X** 
X** 
TMS side effects questionnaire 
 
X 
X 
X 
Baseline fcMRI/FDG-PET without rTMS 
X 
 
 
 
fMRI before and after rTMS; FDG-PET after rTMS 
 
X 
X 
X 
 
**Exact frequency of pregnancy testing will vary according to how far visits are apart 
 
Table 1: Summary of study procedures by visit 
 
VI. BIOSTATISTICAL ANALYSIS 
fcMRI data: The main outcome measures of interest for fcMRI data will be the relative change in z-transformed 
correlation coefficients between two given cortico-limbic network regions of interest (ROIs), the difference 
between changes in these z-scores across conditions (e.g. correlated vs. anticorrelated stimulation), and this 
difference across subjects (e.g. MDD vs. controls). Six cortico-limbic regions of interest will be examined: DLPFC 
(the 2 stimulation sites), sgACC, dmPFC, NA and amygdala.  Seed placement for sgACC will be based on MNI 
coordinates from other fcMRI studies.21,22 Anatomical location of other seeds will be determined on an 
individualized basis as the points of maximal  positive correlation with sgACC (or negative correlation in the case 
of one of the DLPFC sites). Seeds will be spherical and 8mm3 in diameter. The primary correlation pair of 

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interest will be DLPFC-sgACC. Based on prior studies,1-9,30 secondary correlation pairs of interest will be sgACC-
dmPFC, sgACC-NA and sgACC-amygdala. fcMRI data will be analyzed using a combination of software 
packages (e.g., FMRIB Software Library, FSL, Freesurfer, SPM) and custom software developed in the Buckner 
(mentor) lab. These methods have been described in several other paradigms27-29,76,77 and were employed in 
Eldaief et al.14 We will apply a stringent in-house data quality control program to each dataset, described Yeo et 
al.,78 to evaluate fMRI runs for low signal-to-noise and/or excessive head movement. Preprocessing will include 
global mean regression and temporal band-passing of data for signals >0.08 Hz. After preprocessing, region-to-
region correlation strengths will be calculated with volumetric seed-based functional connectivity analyses: 
correlation maps will be produced by extracting the BOLD time course from a “seed” ROI in the brain, and then 
computing the Pearson’s correlation coefficient (r) between that time course and the time course from all other 
brain voxels. Functional connectivity between two ROIs will be measured by r values, which will be transformed 
to the z distribution. Paired t-tests will directly compare changes in z scores before and after rTMS in a given 
individual, and two-sample paired t-tests will compare changes across conditions and across subjects.  
 
FDG-PET data:  MR-based motion estimates and a dual-echo ultrashort echo time MRI sequence (DUTE)79 
will be used for motion- and attenuation-correction of PET data, respectively. Other preprocessing steps will 
include image realignment, normalization to MNI space and smoothing. Weighted linear contrasts will then be 
used to identify changes in rCMRglu in the cortico-limbic regions of interest described above. Relative 
differences in normalized rCMRglc in these ROIs between the baseline PET scan and the post-rTMS PET 
scan within a given individual will be calculated on a voxel-by-voxel basis using a paired t-test.  Two sample 
paired t-tests will compare rCMRglu changes across rTMS conditions, and across MDD patients and normals. 
The experimental PET and fcMRI data revealing local and distributed impacts of TMS will also allow for 
computational analyses which will be developed with the supervision and guidance of Dr. Deco (advisor). 
 
Experimental Controls: To ensure that the effects of rTMS are specific to the cortico-limbic systems 
investigated, we will construct three extra-network seed regions: primary motor cortex (M1), primary auditory 
cortex (A1) and primary visual cortex (V1). A lack of rTMS induced changes in rCMRglu in these regions; and a 
lack of rTMS induced changes in inter-hemispheric functional connectivity between these regions, will 
corroborate network specific effects. To ensure that the effects of rTMS are not the result of inherent variability 
of connectivity between two fcMRI acquisitions separated in time, we will compare pre-rTMS fcMRI scans 
between the two stimulation sessions (correlated and anticorrelated). A lack of significant variability between 
these will argue that changes were caused by rTMS (identical controls were used in Eldaief et al.14) 
Rating scales: Paired t-tests will compare mood analog scale ratings before and after rTMS to assess the 
possibility that stimulation of cortico-limbic networks confers transient mood altering effects. 
 
VII. RISKS AND DISCOMFORTS 
Risks attributable to MRI: There are no known or foreseeable risks associated with conventional MRI 
procedures except to those people who have electrically, magnetically or mechanically activated implants 
(such as cardiac pacemakers), or those who have cerebrovascular clips. Participants will therefore be 
screened very carefully (with a standardized MRI screening form-see attachment) to exclude the possibility that 
they have any such devices and/or implants, and will be excluded from participation in the event that they do. 
There are no known additional risks associated with functional MRI, and the functional MRI systems to be 
employed have been approved by the FDA and will be operated within the standards reviewed and accepted 
by the FDA. All studies will specifically adhere to these FDA approved safety levels for the Siemens system. 
These safety parameters include static magnetic field, time varying magnetic fields (dB/dt), specific absorption 
rate (SAR), and acoustic noise levels. Subjects will be informed about minimal risks of routine high magnetic 
field and non-ionizing RF radiation involved in MR imaging. Still, an MRI might be uncomfortable to a subject 
due to the risk of a) claustrophobia; b) boredom from lying still for an extended time or c) being perturbed by 
scanner noise.  
Risks attributable to TMS: TMS has been used in a growing number of laboratories worldwide since 1984. 
Guidelines for the safe use of rTMS were published in 1993 by Pascual-Leone et al., and were updated in a 
recent paper by Rossi et al. 2009.69 In this paper, parameters for the maximum repetitive TMS train duration, 

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frequency, and intensity in order to safely apply rTMS were described. The most worrisome side effect of TMS 
is that of a seizure, however, this is an extremely rare occurrence in subjects who do not have epilepsy, or who 
are not prone to seizures. To date, very few patients have had seizures associated with TMS reported in the 
literature, among the many thousands of normal subjects who have received it since 1984. In a review of 
published studies that applied rTMS to non-motor areas in healthy participants and patients between 1998 and 
2003, only two seizures occurred among 3092 subjects exposed to rTMS. The precise risk ratio is uncertain, 
but the overall risk for this complication is thought to be less than 1/1,000 studies. TMS has also been rarely 
associated with pseudoseizures and syncope. More common risks associated with TMS, and measures taken 
to limit these, are presented in Table 2.  
 
In terms of seizures, all rTMS sessions will be conducted by the PI (Dr. Eldaief) who is (1) a board certified 
neurologist, (2) has been trained in the safe and efficient administration of TMS (through a course offered at 
the Berenson-Allen Center for Non-Invasive Brain Stimulation at the Beth Israel Deaconess Medical Center) 
and (3) is trained in basic life support and in the recognition and treatment of convulsions, syncope and other 
medical/neurological emergencies. In addition, a fully equipped and regularly checked crash cart will be 
available at all sites where TMS stimulation will occur. This emergency equipment includes oxygen supply, IV 
line supplies, and emergency medications (e.g. benzodiazepines) in the event of a convulsion. Therefore, 
should any complication occur, rapid medical coverage for the subject will be provided, followed by Emergency 
Room care if appropriate. We will also monitor patients in detail during and after delivery of rTMS, using an 
approach drawn directly from suggested guidelines.68 Notably, there is not expected to be an increase in the 
risk of seizures with stimulation of the control site. In fact, stimulation of the cerebellum is much less likley to 
result in seizure induction. Other measures to minimize risks from TMS are listed in Table 2. 
 
Risk Associated with TMS 
Measures taken to prevent/minimize this risk 
20-40% 
of 
subjects 
experience 
MILD 
headaches or neck pain 
Care will be taken to administer stimulation in a 
comfortable neck position and to avoid unnecessary 
stimulation of neck/scalp muscles. 
TMS produces a loud clicking sound which 
may cause tinnitus or rarely short-term hearing 
loss  
Subjects will be given earplugs, which, in animal and 
human studies, proven effective in preventing the risk of 
hearing disturbance due to TMS. 
TMS could induce short-term changes in 
memory, attention and other cognitive functions 
This is a theoretical risk, as none of the safety studies 
conducted has found such side effects. 
Acute psychiatric effects such as mania and 
delusions have been described in patients with 
medically refractory depression or bipolar 
disorder who received rTMS. This seems to be 
a rare complication of rTMS (incidence <0.15% 
of patients). 
Mania and delusions arising from TMS have not been 
described in purely schizophrenic cohorts, e.g. those 
without a comorbid affective disorder. Subjects will undergo 
a brief rTMS side effects questionnaire after rTMS to 
assess for these rare effects. 
Concentration/memory 
deficits 
which 
are 
exceedingly scarce. 
Subjects will undergo a brief questionnaire after every 
rTMS session to assess for these rare effects. 
Table 2. Risks associated with TMS and measures to prevent them 
 
Risks Attributable to PET:  
a) Venipuncture: An intravenous catheter will be placed for this study to inject the FDG radioligand.  FDG 
injection will be administered by a licensed nuclear medicine technologist. All safety procedures related to 
venipuncture (e.g. minimization of bleeding, bruising and infection) will be strictly adhered to and 
venipuncture will be carried out by an experienced technician. As such, subjects are expected to feel 
transient discomfort at the time of venipuncture. There is also the risk of slight bruising at the sight of 
venipuncture. Bleeding or infection risks (e.g. cellulitis) are possible following IV placement, but these are 
exceedingly rare.  
b) Radiation exposure: The radiation exposure in this study will be small and there is no evidence that it 
represents a major health risk. We will follow the safety standards put forth by the Radioactive Drug 

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Research Committee (RDRC) and the Massachusetts General Hospital Radiation Safety Committee. As is 
the convention for receiving FDG-PET, it will not be the total number of scans a subject has, but the total 
amount of radiation exposure per year that will serve as the limiting factor. According to RDRC policy, 
adult research subjects should be limited to a total of 50 milliSieverts (mSv) of radiation dose per year. It is 
estimated that each FDG scan results in a whole body radiation exposure of 3.5mSv per scan. This would 
amount to a total of 10.5mSv of exposure per year for the majority of  subjects (who will each undergo 
three FDG-PET scans) and 14mSV of exposure per year for subjects who agree to undergo an optional 
fourth study visit.  Notably, exposure is limited to this amount because scanning will occur on an MRI-PET 
scanner, and not a CT-PET scanner. Careful monitoring of a subject’s recent radiation exposures will 
occur, and subjects will be prohibited from participation if they have received radiation for any other study 
in the past 12 months. If subjects have participated in other research studies in the past 12 months that 
have involved radiation exposure, they will be asked to inform the investigators or study staff (by placing a 
check mark on the consent form verifying that they have or have not been exposed to other radiation in 
the past 12 months).  If it is determined that the prior radiation exposure exceeds our current guidelines 
(i.e. 15 mSv/year) the subject will not be enrolled in this study.  
 
We will use FDG purchased through commercial sources, specifically through PETNet and Cardinal 
Health. We will follow the safety standards approved by the Radiation Safety Committee for the use of 
radioligands. The IV injection will be administered either by a physician or by a trained technician. There is 
an extremely small risk of allergic reaction to FDG.  Should there be an adverse event, Dr. Eldaief will be 
responsible for communicating with the IRB within the stipulated time frame. 
 
Possible risks associated with combined MRI-PET: There are no foreseeable additive risks from combining 
PET with MRI. The U.S. Food and Drug Administration (FDA) recently gave the first regulatory clearance of a 
hybrid PET/MRI scanner in the U.S.  However, it should be noted that the specific combined PET/MRI machine 
that will be used as part of this study is not FDA approved. 
 
Risks attributable to behavioral testing: The questionnaires employed as part of the study may cause some 
subjects to feel frustrated, bored or upset. It might also cause them to feel dysphoric as it may remind them of 
their clinical depressive symptoms. Subjects will be told that they may stop at any time during the testing.  
 
Possible risks of delaying treatment in MDD subjects: MDD subjects may or may not have a treating 
psychiatric provider during their participation. If the subject does have a current provider, the PI will correspond 
directly with the subjects’ psychiatric provider after seeking formal permission from the subject to do so (in the 
form of a signed medical release form to discuss his/her care supplied by said provider’s office). This will allow 
the PI to (1) discuss maintenance strategies for the subject during their pre-treatment phase, (2) report 
worsening of the patient’s mood to the provider and (3) receive reports from said provider about clinical 
deterioration. Maintenance strategies might include, for example, having MDD subjects undergo brief 
interpersonal therapy (IPT), either through the outpatient Psychiatry department at BWH or directly with their 
provider.  
 
However, in some cases an MDD subject may refuse to have a psychiatric provider and refuse to undergo 
treatment for their depression (even after the risks of doing so are explained to them by the PI). That is, they 
may have decided to not pursue pharmacotherapy or psychotherapy to treat their depression. In these cases, 
treatment would not be “delayed,” as the subject never intended it to be started. For these subjects, the PI (a 
board certified psychiatrist) will still monitor MDD subjects in the following ways to assess symptom worsening 
during their study participation. First, he will directly ask subjects at each of their three visits about possible 
symptom worsening. Secondly, and more objectively, this will be assessed by performing a HAMD 
questionnaire at each of the three study visits. Third the PI will provide brief patient education on Major 
Depression, specifically with respect to common symptomatology, theories as to the biological basis of the 
disorder and treatment options. Fourth, the PI and will encourage that they seek treatment in the form of 
psychotherapy during the study and with psychotherapy and/or pharmacotherapy after their study participation 
ends.  

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In addition, subjects with MDD who experience a 20 percent increase in their HAMD rating scores during the 
course of the study will be removed from the study and referred to a regular psychiatric clinician for the 
institution of prompt treatment. As mentioned, this will be assessed by performing a HAMD at each of the three 
study visits. 
 
Depressed patients will be evaluated at each study visit with the Columbia Suicide Severity Rating Scale 
(CSSRS) as well as the HDRS. If subjects report active suicidal ideation (SI) on either scale they will be 
removed from the study. Of note, this course of action will be taken if a subject endorses active suicidal 
ideation at any visit, even if their HDRS score has not increased by 20% from their last visit. Suicide risk in 
these individuals will be further probed by examining their CSSRS responses. If the risk of suicide is deemed 
high and/or imminent (e.g., if the subject endorses frequent active SI, has intent and/or plan for self-injury) the 
subject will be immediately referred to the Emergency Department at MGH. In these cases of extreme concern, 
subjects will be taken to the ED with a securtiy escort and may have to be taken against their will. If the subject 
is determined to have SI but to not be an imminent risk for suicide, it will be suggested that they contact the 
Boston Emergency Services Team (BEST), which is jointly operated through MGH and Boston University and 
which is well equipped to provide acute care and to facilitate rapid referrals to psychiatric providers. Information 
on contacting the BEST team will be provided on the MDD resource sheet. Obviously, if the subject has an 
established psychiatric provider, they will be encouraged to contact that individual immediately as well. 
 
VIII. 
POTENTIAL BENEFITS 
 
It is not expected that individuals participating in this study will benefit directly from it, other than through financial 
compensation. Furthermore, MRI scans being used are NOT sufficiently clinically diagnostic to detect pathology. 
However, if a pathological finding is suspected, the subject will be referred for further neurological and/or 
radiological work-up.   
 
This work will better elucidate how intrinsic networks and their dynamics are altered in MDD. It will also assess 
how rTMS, or other neuromodulatory strategies, create the formation of more adaptive configurations. In turn, 
this will pollinate future trials that optimize MDD treatments through selective targeting of intrinsic networks. 
 
IX. MONITORING AND QUALITY ASSURANCE 
 
The Principal Investigator (PI), Dr. Eldaief and his staff will have the responsibility for continually monitoring all 
aspects of the studies, including adverse events (AEs), and for reporting AEs according to the PHRC 
guidelines. Adverse effects will be collected from the start of the experimental protocol to the end of study 
participation. All adverse events, regardless of attribution to rTMS, to MRI scanning or to PET scanning or any 
other study associated procedure or questionnaire, will be collected and recorded using standard adverse 
event forms approved by the Partners IRB. Participants will be asked in an open-ended way about the 
presence of any such events. Also, a standard questionnaire for TMS-related adverse effects (see attachment) 
will be performed in the period after every rTMS session. Intensity of each adverse event will be graded as 
mild, moderate or severe. Events will be medically evaluated where appropriate, including testing and referral.  
 
All study investigators attest that they have read the adverse event definitions provided by the PHRC and that 
they have undergone the required training in the protection of human subjects. The PI understands the 
responsibilities and reporting requirements for the PHRC. Adverse events and unanticipated problems will be 
reported to the IRB per PHRC reporting guidelines. 
 
Descriptions of AEs and unanticipated events which are not AEs will include: detailed descriptions of the 
events themselves, descriptions of whether the event was expected or unexpected, descriptions of the severity 
and frequency of the events, determinations as to whether the event is related or possibly related to study 
procedures, determinations as to whether continuation in the study places subjects at an increased risk of 
harm, and whether changes to the research or other corrective actions are warranted.  
 

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To safeguard confidentiality and the privacy of protected health information, each study subject will be 
assigned a unique code number. A separate file linking the patient’s name with study number and identifiers 
will be kept in a password-protected data file, accessible only by the study investigators. All study forms will be 
kept in secure locked file cabinets. The study investigators will assume full responsibility to maintain the 
confidentiality of all data. All study results will be presented only as statistical aggregates that will neither 
identify, nor permit identification of, individual subjects. 
 
Dr. Eldaief, the principal investigator, will review the accuracy and completeness of the data and adherence to 
the PHRC approved protocol on a continuing basis. Guidelines regarding Data and Safety Monitoring Plans 
and Quality Assurance and Adverse Event Reporting Guidelines will be followed at all times. 
 
 
 
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