Physiological and Cognitive Biomarkers for Ketamine's Antidepressant Effects
Phase: NA
Conditions: Depression
Sponsor: Massachusetts General Hospital

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Psychophysiological and Cognitive Biomarkers of Ketamine’s Antidepressant Effects 
(Protocol 2015P002397) 
 
NCT02669043 
 
Version Date: 12/13/2016 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Detailed Protocol: 
 
I. Background and Significance 
 
Establishing remission of major depressive disorder (MDD) often takes weeks to months to achieve on 
traditional monoamine-based antidepressants. Only about one-third of individuals with MDD remit on an initial 
course of treatment, with even lower rates of remission among individuals with certain depressive subtypes, 
such as anxious depression. There is a compelling and widely acknowledged need for antidepressants that 
work more rapidly, that recruit novel, non-monoamine mechanisms, and that target subtypes of depression that 
respond poorly to current antidepressants. Towards this end, single subanesthetic infusions of ketamine (an 
antiglutamatergic medication) have been shown to improve the symptoms of depression in a rapid (within 
hours), robust (across many symptoms), and relatively sustained manner in patients with treatment-resistant 
depression (TRD).(1) Ketamine may have greater efficacy for forms of depression not well treated by current 
agents—especially anxious depression.(2) Low dose parenteral ketamine has now been studied for difficult to 
treat depressive disorders by numerous research groups and off label administration of ketamine is 
increasingly available in some clinical settings.  
 
There is a widely recognized need for developing biomarkers that may predict response to rapidly 
acting antidepressant agents such as ketamine. The identification of biomarkers of treatment response to 
rapidly-acting therapeutics for depression is likely to advance targeted drug development and the goal of 
personalizing depression treatment. The current protocol is aimed at further delineating potential biomarkers of 
ketamine’s effects among individuals with treatment-resistant anxious depression, as well as using putative 
biomarkers to diagnose depression subtypes (i.e., anxious depression).  
 
Background and Preliminary 
Results: Ketamine and Anxious 
Depression. This protocol stems from 
earlier work by the PI with colleagues 
at the NIH/NIMH on the definition,(3) 
neurobiology,(4) and treatment(5) of 
anxious depression. Because anxious 
depression is a common depressive 
subtype, we sought to examine the 
extent to which symptomatically-
diagnosed anxious depression 
predicted response to ketamine. 
Specifically, in our post hoc analysis, 
we found that patients with anxious 
depression (n=15) had significantly 
greater antidepressant responses 
(defined as a ≥50% improvement in 
Montgomery-Asberg Depression Rating 
Scale (MADRS) scores from baseline) 
to ketamine compared to patients with 
nonanxious depression (n=11).(6) This 
effect was observed over the course of 28 days (see Figure 1; * = p<0.05), with the largest effect at Day 2 
(Cohen’s d=0.76)—a clinically relevant finding, given that anxious depression is typically more difficult-to-treat 
than nonanxious depression.(5) In addition, patients with anxious depression had significantly longer median 
days-to-relapse compared to nonanxious depression patients (19.0±17.9 vs 1.0±0.0 days-to-relapse, 
respectively; p=0.002).The two groups did not differ in their experiences with the dissociative (p=0.62) or 
psychotomimetic (p=0.41) side effects of ketamine. These results suggested that symptomatically-diagnosed 
anxious depression may be a clinical predictor of ketamine response, a particularly intriguing finding in the 
context of the majority of clinical trials involving standard antidepressants in which anxious depression is 
related to poorer antidepressant response.  

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Though symptomatically-diagnosed anxious depression appears to predict a better antidepressant 
treatment response to ketamine compared with depression without anxious features, several important aspects 
remain to be elucidated include: 1) the neural mechanisms through which ketamine exerts its superior 
antidepressant effects in anxious depression, 2) the relationship of objective and subjective measures of 
anxiety within mood dysregulated patients (Aim #1), and 3) the extent to which objective physiological 
measures of anxiety might predict patients’ response to ketamine (Aim #2). One theory that may help to 
explain ketamine’s superior antidepressant properties in anxious depression compared to nonanxious 
depression involves the reward/motivation brain circuit. The combination of reward pathway dysfunction 
and hyperactive threat responses has been suggested as a mechanism for the pathophysiology of anxious 
depression.(7, 8) Indeed, anxiety, depression, and dysfunctional reward processing are highly comorbid.(9) 
Furthermore, a recent review of the literature(8) highlighted that chronic stress (a model of anxiety), aversive 
stimuli, and punishment all lead to “stress-induced” clinical symptoms of anhedonia, low motivation, and 
depression through indirect inhibition of the reward circuitry.  
The reward circuitry receives both excitatory (glutamatergic) and inhibitory (GABAergic) inputs from the 
bed nucleus of the stria terminalis (an area of the brain involved in hypervigilant threat monitoring and 
anxiety).(10, 11) Preclinical research has suggested that during aversive events, the reward circuitry is 
indirectly inhibited by increased excitatory (glutamatergic) and decreased inhibitory (GABAergic) neuronal 
activity from the bed nucleus of the stria terminalis.(12) Conversely, a reduction in anxiety-related behaviors 
and an increase in reward-motivated behaviors are observed when this inhibitory tone is increased.(11) 
Altogether, these findings suggest that the bed nucleus of the stria terminalis may have a unique role in 
integrating stress information with reward responses.(13)  
Ketamine, an N-methyl-D-aspartate (NMDA) receptor glutamatergic antagonist, may work in the 
treatment of anxious depression by blocking actions from the excitatory (glutamatergic) neurons in the bed 
nucleus of the stria terminalis, thereby blocking the “stress-induced” inhibition of the reward circuitry. In 
depressed patients, this action may reinstate more normal functioning of the reward circuit. Indeed, in one 
recent mouse study, it was shown that knocking out the glutamatergic receptors within the bed nucleus of the 
stria terminalis mimicked the antidepressant effects of systemic ketamine administration, further implicating the 
bed nucleus’ relationship with ketamine’s antidepressant effects.(14) We propose several noninvasive 
experiments to interrogate these pathways.  
 
Background and Preliminary Results: Psychophysiology and Cognitive Testing. In this protocol, 
several psychophysiological and cognitive experiments will be used to objectively measure anxiety for the 
prediction of ketamine’s antidepressant effects. The startle eye-blink reflex is a well-documented primitive 
defensive/protective reaction that occurs when an individual is exposed to an unexpected, mildly aversive 
stimulus such as a highly annoying but not painful electric shock administered (15). In the literature on the 
startle reflex, a distinction has been made between fear and anxiety. Fear is thought to be associated with 
predictable and phasic administration of an aversive stimulus while anxiety is thought to be associated with an 
unpredictable and sustained aversive stimulus. Despite their clinical similarities, assessing both fear- and 
anxiety-potentiated startle response is critical, as a growing literature suggests that their pathophysiology 
involves distinct brain circuits—specifically, phasic fear-potentiated startle is mediated by the amygdala, 
whereas sustained anxiety-potentiated startle is mediated by the amygdala and the bed nucleus of the stria 
terminalis.(15, 16)  
In previous work, we used the NPU-threat test (No shock, Predictable shock, and Unpredictable shock) 
to examine whether baseline fear- and anxiety-
potentiated startle (as elicited by electric shocks) differed 
between a heterogeneously diagnosed group of patients 
with depression (n=28) and healthy volunteers (HV; 
n=28).(17) Briefly, participants intermittently experienced 
abrupt bilateral auditory stimuli (40-ms duration, 103-dB 
noise burst) through headphones, eliciting a startle 
response. These startle responses were then measured 
during experiments of predictable (signaled by a cue) and 
unpredictable (unsignaled) shock, related to fear and 
anxiety, respectively. Baseline startle responses revealed 

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significantly greater startle magnitude in the depressed group compared to the HV group (p<0.02; Figure 2), 
extending both to fear- and anxiety-related potentiation. Furthermore, the depressed group exhibited significant 
increases in startle upon the mere placement of the shock electrodes (reflecting contextual anxiety; p<0.005) 
compared to a period prior to shock electrode placement—a difference that was not seen in the HV group. 
In addition to eye-blink startle, we will collect other physiologic measures, including autonomic 
responses to threat stimuli that have a demonstrated link to pathological anxiety in other studies. Specifically, a 
larger heart rate response to loud tones (a threat stimulus) has been shown to be one of the most robust 
physiological findings in patients with current PTSD, and may reflect increased defensive responding to 
threats.(18-21) This increased reactivity appears to be acquired after the onset of the disorder, rather than pre-
existing, further suggesting that this physiological marker is an accurate indicator of disease.(18) Heightened 
heart rate activity has also been observed in patients with obsessive compulsive disorder.(22) It is our 
hypothesis that autonomic responses to threat stimuli will be similarly found as indicators of anxiety within 
depression. 
As described earlier, dysfunction in reward processing may be critical in the pathophysiology of anxious 
depression (7,8). In order to examine the spectrum of reward dysfunction in depressed patients, as well as to 
test the extent to which cognitive deficits in reward processing predict ketamine’s antidepressant effects, we 
will conduct a pre-treatment probabilistic reward task, an objective measurement of participants’ ability to 
modulate behavior as a function of rewards. Clinically, depressed patients have a deficit in reward learning (a 
construct of the so-called RDoC [Research Domain Criteria] positive valence domain), as they have difficulty 
modifying their behavior despite positive reinforcement.(8) In one previous probabilistic reward task study, pre-
treatment inability to modify reward learning in depressed patients predicted continued depressive symptoms 
at 8-weeks after treatment was initiated.(23) In view of ketamine’s effect on the NMDA receptor and its 
relationship to reward processing, we hypothesize that greater reward dysfunction will predict response to 
ketamine.  
In summary, we found that subjectively-diagnosed anxious depression predicts a greater 
antidepressant response to ketamine compared to nonanxious depression. Aberrant brain circuitry between 
the bed nucleus of the stria terminalis and the reward circuitry may contribute to the psychopathology observed 
in the anxious-depression phenotype, and might be the targets that account for ketamine’s superior 
antidepressant efficacy among individuals with this depressive subtype. Therefore, higher pre-treatment 
psychophysiological (i.e., anxiety-potentiated startle magnitude) and cognitive (i.e., reward dysfunction) 
measures of anxiety in depressed patients may serve as biomarkers that can clinically predict better treatment 
response to ketamine and that reflect the activity of specific circuits that could be targeted for future drug 
development. 
 
 
II. Specific Aims 
 
The overall aims of this protocol are to study biomarkers for diagnosis and treatment response in mood 
disorders. Specifically: 
 
Specific Aim #1: Determine the extent to which objective measures of anxiety are related to 
subjective measures of anxiety in patients with mood dysregulation. Our working hypothesis is that 
patients with clinically-diagnosed anxious depression will have higher objective measures of anxiety (anxiety-
potentiated startle responses and lower reward responsiveness) compared to those with nonanxious 
depression. 
 
Specific Aim #2: Determine the extent to which these biomarkers predict response to ketamine. 
Our working hypothesis, based on pilot data and previous work, is that higher pre-treatment 
psychophysiological measures of anxiety-potentiated startle and lower reward responsiveness on cognitive 
measures will correlate with a better antidepressant response to ketamine.  
 
Specific Aim #3: Determine the effect of ketamine on the biomarkers. Our working hypothesis is 
that greater reductions in anxiety-potentiated startle magnitude, and greater improvements in measures of 
reward responsiveness, will be found among ketamine responders compared to nonresponders.  
 
III. Participant Selection 
 
Inclusion Criteria: Patients 

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Patients will:  
1) be 18-64 years old,  
2) read, understand, and provide written informed consent in English,  
3) meet criteria for a primary psychiatric diagnosis of Major Depressive Disorder (MDD) for ≥ 4 weeks, 
according to the Mini International Neuropsychiatric Interview (M.I.N.I.) and have a Hamilton Depression 
Rating Scale (HDRS-28) total score ≥ 20; depression may have started at any time point in their life, and 
certain co-morbid diagnoses (e.g., anxiety disorders) will be allowed,  
4) have a history ≥1 failed medication trial during the current depressive episode (per the MGH 
Antidepressant Treatment History Questionnaire),  
5) be on a stable adequate dose of an FDA-approved antidepressant medication for ≥28 days prior to 
Study Phase II (see medication inclusion/exclusion list),  
6) maintain a treating doctor who is in agreement with study participation,  
7) have a reliable chaperone to accompany them home following the completion of Visit 3 (the 
ketamine infusion day),  
8) be generally healthy, as assessed by medical history, physical examination (including vital signs), 
clinical laboratory evaluations, and electrocardiogram (EKG), 
9) be of non-childbearing potential or use of an acceptable form of birth control (females only). 
 
Exclusion Criteria: Patients  
Patients will be excluded if any of the following criteria are met:  
 
1) delirium or dementia diagnosis,  
 
2) unstable medical illness or clinically significant laboratory results,  
 
3) history of clinically significant cardiovascular disease or electrocardiogram (EKG) findings, or medical 
conditions that put the patient at risk for possible cardiac side effects (e.g., requirement of cardiac pacemaker) 
or alter brain morphology (e.g., recent head trauma, post intracranial surgery, intracranial mass or bleed or 
unstable sleep apnea), or a blood pressure >140/95 mmHg at Screening, 
 
4) history of multiple adverse drug reactions, (e.g., history of hives or anaphylaxis in response to a 
medication, severe intolerance and/or severe side effects to a medication), including hypersensitivity to 
ketamine, 
 
5) current/past history of psychotic disorders, history of out-of-body feelings or derealization, 
 
6) active substance use disorders (except nicotine and caffeine) within the past six months or past 
history of ketamine/PCP abuse (we will confirm this with collateral information from their doctor if necessary), 
 
7) requirement of excluded medications  that may interact with ketamine (see exclusionary medications 
list), 
 
8) caffeine or nicotine use within 1 hour of psychophysiology testing, or alcohol use within 1 day of 
testing, 
 
9) pregnancy, breastfeeding, or unacceptable means of birth control (females only) 
 
10) clinically significant hearing impairment, 
 
11) current serious suicidal or homicidal risk,  
 
12) concurrent participation in other research studies involving medications or other treatments, 
 
13) narrow angle glaucoma, 
 
14) acute intermittent porphyria history, 
 
15) history of seizures in the past 6 months, regardless of seizure type, 
 
16) hyperthyroidism or untreated hypothyroidism,  
 
17) airway instability or pulmonary disease with hypercarbia, or 
 
18) current or past cubital or carpal tunnel syndrome. 
 
IV. Participant Enrollment 
We will enroll 58 depressed patients outpatients into the study. 
Recruitment: 58 outpatients will be enrolled via response to flyers, advertisements, e-mails, 
participation in previous studies with an interest in participating in further research, and letters to providers. The 
DCRP has used these methods for over two decades with considerable success, receiving an average of over 
35 phone calls/week.  

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Retention: All patients are eligible to receive a free consultation at the DCRP immediately following the 
completion of all study materials. This care is supplemental to their ongoing care with their treating 
psychiatrist/doctor. The treating psychiatrist will be informed before any medications changes are considered.  
 
Renumeration: All participants who complete all parts of the study will be paid $200, and parking will be 
validated for the study visits. For patients that come in for the screening visit only, they will be renumerated 
$20; parking will also be validated. The payment schedule for the other three visits is $60 each.  
 
Note, the probabilistic reward task is a monetary reward task. Patients will be told that their payment for 
this task, based on the money they win, will be included in their total payment for study participation.  
 
Procedures for Informed Consent 
Written informed consent will be obtained by a licensed physician from all patients before protocol-
specific procedures begin. The investigator obtaining consent will explain in detail the protocol of the study, its 
purpose, and potential benefits to the society. Participants will be informed that they can choose to terminate 
the study at any time, for any reason.   
 
Treatment Assignment and Randomization (If Applicable) 
 
N/A; All participants will be assigned to open-label ketamine. 
 
V. Study Procedures 
 
Research Design Summary: The proposed research (Aims 1-3) will begin with a screening visit (Visit 1) 
and a medication stabilization period of ≥ 28 days (Phase I). In Phase II, 58 depressed patients meeting 
research criteria (specified above) will undergo baseline biomarker and psychometric assessments (Visit 2). 
One-to-two days later, they will receive an open-label infusion of subanesthetic intravenous ketamine 
(0.5mg/kg over 40 minutes), and will be monitored for 4 hours in the MGH Clinical Research Center (Visit 3). 
Visit 4, which will occur on the day after the ketamine infusion, will consist of the same biomarker and 
psychometric assessments from Visit 2 (see “Schema” for overall study outline): 
 
 
 
 
 
 
 
 
 
 
Visits and Parameters to be Measured/Data to be Collected and When the Data is Collected 
Study Phase I: Screening: At Visit 1 (“Screening” visit), patients will undergo initial evaluation (i.e., 
physical and psychiatric examination, vital signs, blood genetics and biomarkers collection, neuropsychological 
testing, basic laboratory and pregnancy/urine toxicology testing, EKG, hearing assessed for the ability to detect 
a white noise stimulus at 25dB) following informed consent to evaluate their eligibility based on 
inclusion/exclusion criteria. This will occur at the MGH DCRP. Prior to Study Phase II, patients will remain 
stable on their antidepressant dose for ≥28 days.  
In order to ensure that participants do not have a history of substance abuse or dependence, we will 
perform a structured clinical interview, as well as urine toxicology screen at Visit 1. Furthermore, if necessary, 
we will obtain permission from participants to contact their primary physician or other treaters, to obtain further 
collateral information about substance use problems. 
Study Phase II: Testing and Treatment: Study Phase II consists of three visits (Visits 2-4) over four 
days. At Visit 2, participants will have pre-treatment psychophysiological and cognitive testing assessments in 
the morning. Visit 3 will occur ideally 24 hours after Visit 2, (but within 1-2 days), and will consist of an infusion 
of ketamine (0.5mg/kg over 40 minutes). Prior to ketamine administration, participants will have a urine test for 
pregnancy (females only). Intravenous ketamine will be administered at the Clinical Research Center by a 
medical doctor. Participants will be monitored for 4 hours after the start of the infusion at the CRC trained 
Visit 1: Screening
Visit 2: Baseline biomarker 
and psychometric 
assessments
Visit 3: ketamine infusion
Visit 4: Final  biomarker and 
psych assessments
Phase I: ≥ 28 
Phase II:  ≤4d 

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research nursing staff. Vital signs (Temp, Pulse, Respiration Rate, BP, SpO2 and Sedation level) will be 
measured at +0 minutes, +5, +10, +15, +20, +30, +40, +60, +90, +120, +180 minutes in relationship to the time 
of ketamine infusion.  At the +240 minute mark, participants will have a post-ketamine blood sample drawn for 
genetics and biomarkers. Afterwards, participants will be discharged home into the care of a responsible adult 
escort.  At Visit 4 (which is 24 hours after Visit 3), participants will undergo final psychophysiology and 
cognitive testing, identical to those from Visit 2, as well as have a blood sample drawn for genetics and 
biomarkers. All visits in Phase I and II will include psychometric assessments. 
 
Safety and Monitoring:  
 
Special Case of Sertraline and the Urine Drug Screen  
Sertraline (Zoloft) is known to cause frequent false positives on urine drug screens for benzodiazepines 
(see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2728940/).  Over the past several years, the DCRP has 
experienced an increase in false positive urine drug screens for benzos while patients were taking sertraline. In 
the case when a patient (who is taking sertraline for their depression) is screened for the study and tests 
positive for benzodiazepines on urine drug screen (despite denial of benzo use), the following precautions will 
be taken: 1. The patient's physician will be contacted for collateral information pertaining to alcohol and 
substance abuse, including benzodiazepine use; 2. The patient will be checked in the Mass PAT system for 
active benzodiazepine prescriptions within the past year from the screening date. If the patient's physician 
confirms that they are not taking benzos, and there is no evidence of benzo use in Mass PAT, the patient will 
be deemed eligible for the study for this particular criteria. As a precaution, the patient will be informed that if 
they are, in fact, taking benzodiazepines, they may interfere with ketamine's mechanism of 
antidepressant/antisuicidal action, rendering ketamine less effective. Furthermore, the concomitant use of 
benzos and ketamine may lead to more severe side effects (such as increased somnolence) when combined.  
 
Special Case of Medical Marijuana Use   
Ketamine and Cannabis: Marijuana was recently passed for legal medicinal use in the Commonwealth of 
Massachusetts. As a result of this law, the occurrence of medical marijuana prescriptions is likely to increase. 
Subjects who indicate use of medical marijuana will be reviewed on a case-by-case basis by the PI, and will 
not be immediately excluded from the study. The PI will speak with the participant’s physician and confirm that 
the medical marijuana is prescribed for medical purposed; furthermore, the physician will be asked to confirm 
that the subject does not have a history of substance or alcohol misuse. This exception will only be given to 
participants who are currently prescribed marijuana by a medical professional and will not apply to those who 
use marijuana recreationally.  
MONITOR: Central nervous system- and/or respiratory-depressant effects may be additively or synergistically 
increased in patients taking multiple drugs that cause these effects, especially in elderly or debilitated patients. 
MANAGEMENT: During concomitant use of these drugs, patients should be monitored for potentially 
excessive or prolonged CNS and respiratory depression. Patients will be counseled to avoid hazardous 
activities requiring mental alertness and motor coordination until they know how these agents affect them, and 
to notify their physician if they experience excessive or prolonged CNS effects that interfere with their normal 
activities. 
 
Ketamine Administration and Monitoring: A single, open-label infusion of ketamine at a subanesthetic 
dose of 0.5mg/kg over 40 minutes will be administered on the morning of Visit 3 at the MGH Clinical Research 
Center (CRC) by a BLS and ACLS-certified licensed physician, as this dose and procedure has been 
previously used in depression research, (1, 24, 25) including studies completed at the MGH CRC (e.g., 
Protocol 2012P001042). Prior to the infusion, all patients will be fasting since midnight, except for required 
morning medications. Monitoring of oximetry, pulse rate, and blood pressure will be done before, during, and 
after the infusion. Although subanesthetic ketamine has been shown to have a favorable safety profile when 
given to depressed patients in research studies,(1, 24-27) there is a potential risk of adverse psychotomimetic, 
dissociative, and/or sympathomimetic events. Patients will be discontinued from the study if they experience 
serious adverse events during or after the infusion. See below for details on monitoring and rescue 
medications.  

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Psychophysiology Testing: At Visits 2 (“pre-ketamine”) and 4 (“post-ketamine”), psychophysiological 
testing will be used to measure variations in startle responses (elicited by auditory white noise stimuli) during 
three conditions: no-shock (N), predictable (P) shock, and unpredictable (U) shock (“NPU-threat test”).(28) The 
NPU threat test has been approved for the use in several protocols by the Human Subject Committee at the 
National Institute of Mental Health/National Institute of Health. Similar shock experiments have been approved 
by the Partners IRB (e.g., “The Psychophysiology of Delayed Extinction and Reconsolidation in Humans,” PI 
Scott Orr, PhD). First, patients will be seated in the testing room. Second, electromyogram (EMG) of the 
obicularis oculi muscle will be recorded using a BioPac EMG amplifier via two electrodes placed under the left 
eye to assess blink magnitude of the startle response; this is an established reliable measure of startle 
magnitude in humans.(29) Third, skin conductance will be measured directly from the non-dominant hand 
using a BioPac EDA amplifier with a constant value of 0.5 V. Fourth, heart rate will be recorded using standard 
limb EKG leads connecting to a BioPac EKG amplifier. Interbeat interval will be measured from the EKG and 
then converted to heart rate. Next, nine 103-dB auditory white noise stimuli will be delivered biaurally via 
headphones every 25-30 sec for the purpose of habituating the study patient to startle response (Habituation 
1). Following habituation, two disc electrodes will be placed on the left wrist for the purpose of administering 
the shocks. Shocks are an aversive stimulus and are intended to be highly annoying, but not painful, with 
intensity not in excess of 80 mA and of 500ms duration. Prior to starting the NPU-threat test, patients will 
experience up to fifteen “sample” shocks for the purposes of 1) choosing an appropriate subjective level that is 
“highly annoying but not painful” (the “shock work-up”), and 2) allowing for withdraw from the study if the stimuli 
are perceived as excessively unpleasant. 
Prior to testing, (females only) will receive a pregnancy screen. For the NPU-threat test, patients will be 
seated at a computer. Text will appear continuously on the computer screen to indicate the cue for the 
upcoming testing condition as follows: “no shock” (N), “shock only during cue” (P), or “shock at any time” (U). 
The cue (represented by a geometric shape) will then be displayed during the testing condition, with cues 
differing in color between N, P, and U, though they are meaningless in the N and U conditions. Each testing 
condition will last for 150 sec, with four cues presented during each condition (See Figure). Cues will be 
displayed for 8 sec each time. Patients will be presented with two blocks of conditions: 1) P-N-U-N-U-N-P and 
2) U-N-P-N-P-N-U. Two shocks (the aversive stimulus) will be administered during each of the P and U 
conditions, for a total of 8 shocks throughout the entire block. Specifically, shocks will be delivered at the end 
of the cue in the P condition and in the absence of a cue in the U condition. No shocks will be delivered during 
the N condition.  
Each individual condition (N, P, U) will have one auditory startle stimulus during three of the four cues 
(1-4 sec following cue onset), and three auditory startle stimuli during cue-free periods (i.e., the intertrial 
interval, or ITI), for a total of six startle stimuli per condition. The mean intertrial interval will range from 25-30 
sec, and we plan to minimize short-term sensitization of startle 
by delivering the auditory startle stimulus (no less than 8 sec 
after the aversive shock stimulus). Following each block, 
patients will subjectively rate their anxiety levels during the cue 
and intertrial interval for each condition (N, P, U) on a written 
scale of 0 (not anxious) to 10 (extremely anxious). Patients will 
also rate their anxiety on a scale from 0 to 10 during ths task, 
using the computer keyboard. Throughout the experiment 
heart rate and skin conductance will be continuously recorded 
with the BioPac systems during administration of the NPU-
threat test at Visits 2 and 4. The data will be preprocessed and 
scored using AcqKnowledge software.  
Cognitive Testing: Cognitive testing will be carried out 
to measure behavior and self-reports of anhedonia, reward 
processing and pattern separation. Regarding behavior, we 
will use a validated 25-minute computer-based Probabilistic 
Reward Task(30) at Visits 2 (“pre-ketamine”) and again at Visit 4 (“post-ketamine”). Patients will be given 
verbal instructions for the task in that the objective is to win as much money as possible, which will be handed 
out in cash at the end of the task. The task will consist of 3200 trials, divided into 2 blocks of 100 trials, with 

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blocks separated by a 30-sec break. Each trial will start with the presentation of an asterisk for 500 msec in the 
center of the computer screen to serve as a fixation point. Then, a mouthless cartoon face will appear in the 
center of the screen. After a delay of 500 msec, either a short mouth (11.5mm) or a long (13mm) mouth will 
briefly appear on the face for 100 msec, and then will disappear. The mouthless face will remain on the screen 
until the patient chooses a computer key to identify which mouth (long or short) was presented. For each block, 
both stimuli will be shown an equal number of times. Correct responses will be reinforced with monetary 
reward feedback that will be given as follows: “Correct! You won 20 cents.” In order to induce a response bias 
toward one of the mouth lengths, an asymmetrical reinforcer schedule will be used; for example, correct 
responses for 1 mouth (e.g., the long mouth) will be rewarded three times more frequently than correct 
responses for the other mouth (e.g., the short mouth). This designation will be randomized across patients. 
Because of this unequal frequency of reward feedback, patients with high reward responsiveness are predicted 
to favor the stimulus with more reinforced positive feedback, whereas patients with low reward responsiveness 
are expected to have a lower bias, or none at all, towards the positive feedback. As in published work(30-32) 
for the task, the main dependent variables will be response bias across blocks as well as Reward Learning [= 
Response Bias (Block 3) - Response Bias (Block 1)].   
In addition, we will include a computer based pattern separation task at Visits 2 and 4. The pattern 
separation task is a high throughput behavioral task that captures the input-output transformation function 
characteristic of pattern separation processes. (33) For example, if you park your car in the same lot everyday, 
but not the same space, pattern separation is thought to be involved in the process of you finding your car 
everyday despite being in a different space; this may be dysfunctional in people with depression. In this task, 
patients are shown a series of every-day objects (e.g., a car, garden tool, food, etc.) and are asked to identify 
the objects as being indoor or outdoor objects. Immediately after this, a second part of the task is started in 
which the patients are shown another series of objects. They are asked to call the objects as “old” if they have 
seen the objects before in the task, “new,” or “similar.” As previously done by Stark and colleagues,(33) a third 
of the objects in the testing phase are “old”, “similar” and “new”. Identifying a “similar’ object correctly conveys 
pattern separation, whereas, incorrectly identifying it as “old” conveys pattern completion. By plotting 
responses as a function of object similarity, we can generate an input-output transfer curve. This task will serve 
the purpose of rapid assessment of putative changes in pattern separation. 
Psychometric Assessments: Self- and clinician-rated assessments will take place during the study. We 
will administer the following scales to all participants at all visits (Visits 1-4): the Hamilton Depression Rating 
Scale (HDRS) (the primary dependent outcome measure) is a 28-item validated clinician-administered scale 
that is widely used as an observational rating measure of depression presence and severity;(34) the 
Montgomery Asberg Depression Rating Scale (MADRS), a 15-item validated clinical-administered scale widely 
used for the assessment of depression (35); Hamilton Psychiatric Rating Scale for Anxiety (HAM-A) is a 14-
item validated clinician-administered scale that is widely used as an observational rating measure of anxiety 
presence and severity;(36) Clinically Useful Depression Outcome Scale (for the DSM-5 Anxious Distress 
Specifier) (CUDOS-A) is a 22-item self-rated scale for the assessment of DSM-5 anxious distress;(37) Young 
Mania Rating Scale (YMRS) is an 11-item clinician-rated scale for the measurements of hypomanic/manic 
symptoms;(38) MGH Sexual Functioning Questionnaire (SFQ) is a 7-item self-rated scale for the assessment 
of sexual functioning; Snaith-Hamilton Pleasure Scale (SHAPS) is a 14-item self-report questionnaire for the 
assessment of anhedonia (please loss to previously pleasurable activities);(39)  
We will administer the following to all participants at Visit 1 only (screening): Mini-International 
Neuropsychiatric interview (M.I.N.I.) to assess for the presence of depression;(40) the MGH Antidepressant 
Treatment History Questionnaire (ATRQ), a clinician-rated scale used to determine treatment resistance in 
depression;(41) the Early Trauma Inventory Self Report-Short Form (ETISR-SF), a 29-item self-report item on 
early trauma history,(42) the EHI (a self-reported measure of dominant handedness), and a demographics 
form.  
We will administer the following scales to all participants during Visit 3 (ketamine administration) only: 
Brief Psychiatric Rating Scale (BPRS) is an 18-item clinician-administered scale for the assessment of 
psychotic symptom constructs;(43) Clinician Administered Dissociative States Scale (CADSS) is a 28-item 
clinician-administered scale for the assessment of dissociative symptoms.(44) BPRS and CADSS will be 
administered at baseline, +40 minutes, +80 minutes, and +120 minutes in reference to the start of the 
ketamine infusion. 

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Study clinicians at the DCRP will be responsible for administering all clinician-administered scales and 
assessments. Clinicians have been extensively trained in the use of the HDRS28 by videotapes and live patient 
interviews.  
Concomitant Medications and Adverse Events: Concomitant medications (dosage, start and stop dates) 
will be reviewed and recorded at all visits in the chart. Adverse events will be recorded at all visits on the 
Adverse Events Form.    
Peripheral Blood Sample Genetics and Biomarkers: At Screening, one 10 mL blood sample, one 8-
10mL blood sample, one 6-8 mL blood sample, and two 10mL blood samples will be collected; two 8-10mL 
blood samples will be collected and at Visits 3 and 4, to obtain DNA for possible pharmacogenetic and 
biomarker (e.g. cytokines, metabolites, etc.) studies. The PI will ensure that appropriate privacy and de-
identification procedures are in place for the collection of biomaterials. 
In order to collect iPS cells for future biomarkers and genetics studies, all subjects will be referred to 
participate in Protocol 2009P000238, “The Use of Human Skin Cells and Blood Derived Cells in the Creation of 
Cellular Models of Neuropsychiatric Disorders” (P.I.: Perlis).” Subjects’ choice as whether or not to participate 
in 2009P000238 will have no bearing on their ability to participate in the current protocol.  
 
Drug to be Used: Ketamine will be the only research drug used as part of the study. 
Ketamine Administration and Monitoring: Ketamine is a glutamatergic receptor antagonist that has been 
administered to millions of patients worldwide as a general anesthetic, though its antidepressant mechanism of 
action (at sub-anesthestic doses) remains largely unknown. Although low-dose ketamine has been shown to 
have a favorable safety profile when given to depressed patients in several studies,(1, 24-27) there is a 
potential risk of adverse psychotomimetic, dissociative, and/or sympathomimetic events. Similar to previous 
studies showing efficacy and safety of intravenous ketamine in adults with mood disorders,(1, 45, 46) including 
studies completed at the MGH CRC (e.g., Protocol 2012P001042),  ketamine will be administered at 0.5mg/kg 
over 40 minutes by a BLS and ACLS-certified, licensed physician at the MGH General Clinical Research 
Center. Patients will be monitored at the General Clinical Research Center by a medical doctor and trained 
nurse for changes in vital signs (i.e., heart rate, blood pressure, respiration, temperature, pulse ox, and 
sedation level) and for treatment-emergent side effects (i.e., as measured by the CADSS and BPRS) during 
administration and post-administration. Rescue medications, including intravenous labetalol, lorazepam, 
ondansetron, and haloperidol will be available in the event that unwanted side effects occur; participants 
requiring such interventions will be withdrawn from the study. PO ibuprofen and acetaminophen will also be 
available. The following parameters are specified in the order set for the CRC: 
 
1. Oxygen 4 to 6 liters/min via face mask or nasal cannula PRN, SpO2 <93% at bedside 
2. Labetolol 2.5 mg IV push over 2 mins PRN BP ≥3 readings in a row of BP ≥ 160/100; may repeat 
every 2 to 5 mins X5. Given by MD/NP. Infusions will be stopped if BP ≥160/00 persists for more than 20 
minutes. PRN for tachycardia >100 bpm; symptomatic tachycardia >100 bpm requires EKG.  
3. Lorazepam 2mg IV push PRN severe agitation, attempting to pull out IV; May repeat every 2-5 
minutes X 3. Given by MD/NP 
4. Haloperidol 1-2mg IV push PRN delirium (not oriented to space and time, hallucinations, attempting 
to pull IV); May repeat every 10 minutes X 5. Given by MD/NP 
5. Glycopyrrolate 0.1mg IV push PRN excessive salivary secretion; May repeat every 2-5 minutes X 3. 
Given by MD/NP 
6. Ondansetron 4mg IV PRN severe nausea, may repeat x1 after 30 min if no effect. 
7. Acetaminophen 325mg tablets, take 1 - 2 tablets PO PRN headache, may repeat x1 after 30 min 
8. Ibuprofen 200mg tablets, take 2 tablets PO PRN headache, may repeat x1 after 30 min 
 
Research staff will be responsible for administering scales and for monitoring side effects.  In order to 
minimize external stimuli, ketamine administration will occur in a room with low lighting and minimal noise 
throughout the administration, as well as during the monitoring period. Participants will be given a small snack 
2 hours after the end of the ketamine infusion.  
 

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Discharge: After the ketamine infusion, if medically stable (as deemed by a board-certified physician 
study investigator), participants will be discharged home with a responsible family member or other adult 
caretaker. Discharge home will be based on criteria established by the MGH Department of Anesthesia 
practices for discharge from the hospital following ambulatory surgery. The participant must have stable vital 
signs, be able to respond appropriately to normal commands, be pain free, be free from any nausea and 
vomiting, and have no bleeding from the intravenous sites. Participants must be able to walk unassisted and 
have an accompanying adult to escort them home. Participants will be advised not to return to work and 
against driving or operating heavy equipment for 24 hours.  
 
Medication Transport and Storage 
 
The MGH Research Pharmacy and MGH CRC will store medications, including ancillary medications.  
 
Devices to be Used 
N/A 
 
Procedures/Surgical Intervention 
N/A 
 
VI. Biostatistical Analysis 
Specific Data Variables to be Collected 
As described above, we will administer the following scales at all visits (Visits 1-4): HDRS, MADRS, 
HAM-A, CUDOS-A, YMRS, SFQ, and SHAPS.  
We will administer the following at Visit 1 only (screening): M.I.N.I., ATRQ, and ETISR-SF.  
We will administer the following scales during Visit 3 only (ketamine): BPRS and CADSS at baseline, 
+40 minutes, +80 minutes, and +120 minutes in relationship to the time of ketamine infusion. 
Vital Signs will be recorded once at Visit 1 (the screening visit), and again at Visit 3 at baseline, +0 
minutes, +5, +10, +15, +20, +30, +40, +60, +90, +120, +180 minutes in relationship to the time of ketamine 
infusion.  
 
Study Endpoint 
 
Primary clinical endpoint is change in the HDRS28 total score. Scores that decrease by ≥ 50% from 
baseline will be considered “response.”  
 
Specific Statistical Methods 
Testing of Hypotheses: Statistics and Data Analysis 
 
Specific Aim #1: Objective and subjective measures of anxiety will be positively correlated with 
each other. A primary analysis will examine Pearson correlations between subjective measures of anxiety 
(i.e., the HDRS Anxiety-Somatization Factor Scale, HAM-A, and CUDOS-A) and eye-blink startle response. 
Similarly, we will examine correlations between subjective anxiety and reward responsiveness. We will analyze 
the data to ensure that linearity exists. If linearity does not exist, we will conduct comprehensive sub-analyses 
to find the best way to characterize the relationship of all data. For example, we will use covariates (e.g., 
clinical descriptors such as treatment failures) to determine their role in creating non-linearity. 
 
Specific Aim #2: Greater psychophysiological and cognitive objective measures of anxiety will 
predict better antidepressant response to ketamine, regardless of subjective depression symptoms. 
We will use a nonlogitudinal linear regression model to assess the extent to which the combination of pre-
treatment psychophysiological (e.g., anxiety-potentiated eye-blink startle) and cognitive reward processing 
(high versus low reward responsiveness) predict the antidepressant treatment response to ketamine (Visits 3 
and 4). Antidepressant response to ketamine will be defined as a ≥50% improvement in HDRS scores (primary 
dependent outcome measure). 
 
Specific Aim #3: In ketamine responders, psychophysiological measurements will show a 
significantly larger decreases in magnitude compared to ketamine nonresponders; greater 
improvements will be seen in cognitive reward processing measures in ketamine responders. We will 
use longitudinal analyses (linear mixed effects random regression models) to analyze the effect of ketamine on 

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the biomarkers. These models account for the correlation of observations from the same individual. For the 
change in startle magnitude post-ketamine, the model will include the change in startle magnitude as the 
outcome, and time (Visit 2 and 4) as the predictor. For the change in reward processing post-ketamine, the 
model will include the change in reward processing as the outcome, and time (Visit 2 and 4) as the predictor. 
The coefficient for the correlations in this model quantifies the rate of change in the psychophysiological and 
cognitive measures associated with a change in depressive symptoms. A coefficient significantly different from 
zero indicates that changes in the clinical measures of depression are associated with changes in 
psychophysiological and/or cognitive measures. We will also perform between-group comparisons of 
measurements for responders vs. nonresponders. All models will be fit using SAS software, α=0.05, two-tailed.  
 
Sample Size Determination:  
 
The sample size for Aims #1 and #2 was calculated using the G*Power Sample Size calculator 
(Version 3.9.1.2; downloaded from http://www.gpower.hhu.de) and was based on findings from a post-hoc 
analysis of the antidepressant treatment response to ketamine in anxious versus nonanxious depressed 
patients, where anxious depression predicted a better response to open-labeled ketamine.(6) The anxious 
depression effect size (Cohen’s d) reached its maximum of d=0.76 on Day 2 post-infusion. With this, for Aims 
#1 and #2, we estimate requiring 58 patients with power >0.8, r=0.35 and an α=0.05, two-tailed, in order to 
reject the null hypothesis that there will be no correlations with baseline psychophysiological and cognitive 
measures between ketamine responders and nonresponders. For Aim #3, a sample size of 58 would allow us 
to detect a moderately-large effect size (Cohen’s d≥0.75) for between-group comparisons. In order to account 
for potential drop-outs, we will recruit 70.  
Expected Outcomes/Results:  We expect that subjective and objective measures of anxiety will be at 
least moderately correlated in patients with mood dysregulation. Further, we predict that patients with higher 
pre-treatment levels of objective anxiety, as measured by psychophysiological and cognitive assessments, will 
have significantly greater antidepressant responses to ketamine, compared to patients with lower pre-
treatment objective anxiety. These results will significantly contribute to the paucity of information regarding 
biomarkers of diagnosis and antidepressant response and provide insight into the anxiety and reward 
neurocircuitry that could contribute to the pathophysiology of anxious depression. 
 
VII: Risks and Discomforts 
Complications of Procedures 
Safety: At any time during the study, participants, family members, and treating psychiatrist will be 
encouraged to contact the principal investigator via phone or pager in the case of adverse events or worsening 
symptoms. The treating psychiatrist will be immediately notified of any concerns.  
 
 
Drug Side Effects and Toxicities 
Ketamine: Ketamine is a relatively commonly used anesthetic in both veterinary and human medicine. 
Although it has a good safety profile overall, ketamine has documented sympathomimetic activity that may 
result in mild to moderate increases in heart rate, blood pressure, and cardiac output, though this activity is 
generally short-lived.(47) Findings from previous research on single- and repeated-dose intravenous ketamine 
for treating depression have provided evidence that the autonomic changes that may occur during the active 
administration (i.e., elevated blood pressure, pulse) return to normal shortly after the infusion stops, with no 
clinically significant sequelae.(48, 49) Other possible side effects reported during ketamine infusion include 
arrhythmia, increased salivation, increased bronchial secretions, horizontal nystagmus, euphoria and 
hallucinations. Nystagmus may persist for a period after the ketamine infusion has terminated. 
Rare side effects are allergic reactions (skin rash), pain at site of injection, increased intraocular 
pressure, ulcerations and inflammation in the bladder (reported in ketamine abusers). Ketamine is a controlled 
substance and has the potential for abuse and dependence, in particular in participants with history of drug 
abuse. Participants with history of substance abuse or dependence in the previous year will be excluded.  
In light of ketamine’s relatively good safety profile, all participants will be medically screened (including 
EKG and vital signs) prior to entering the active treatment portion of the study, to ensure healthy baseline 
general and cardiac functioning. In addition, as ketamine is an anesthetic that may result in respiratory 
depression, participants will be excluded from the study if they require sedatives or opiates. During the 

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administration, a medical doctor will remain in the room with a research nurse, the research assistant, and the 
patient, and vital signs will be monitored every 5 minutes during the 40 minutes of administration. Any 
concerning changes will be managed by the physicians. Criteria for discontinuation include ≥3 readings in a 
row of systolic BP ≥180 mmHg or diastolic BP ≥110 mmHg, HR ≥110 bpm, or symptomatic elevations in blood 
pressure (i.e., new onset headache, chest pain, or shortness of breath). Furthermore, the physician may 
choose to stop the infusion at any time if he/she believes it is in the best interest of the participant, based upon 
his/her clinical judgment.  
Participants will stay at the General Clinical Research Center (GCRC) for up to 4 hours following the 
start of the administration, and will be monitored for psychotomimetic, dissociative, and sympathomimetic side 
effects during this time.  Afterwards, they will be discharged home in the care of a responsible adult family 
member or caretaker after completing rating scales with the study physicians. If participants experience 
adverse events at home, they will be referred for evaluation at the nearest emergency department. These 
instructions will also be provided to the responsible adult escort prior to discharge. The participant’s individual 
insurance plan will be responsible for covering this visit.  
Prior to informed consent, potential participants will be informed of alternatives to research, and will be 
provided with the option to seek further psychopharmacological or psychotherapy-based interventions. 
 
Electric Shocks 
The electric shocks that participants receive will be self-selected to be highly annoying, but not painful. 
Subjects may stop the electric shocks at any time. The stimulator is manufactured by Digitimer. It is inspected 
annually by biomedical engineering under the auspices of Partners Human Research Committee (PHRC), who 
have affirmed compliance with safety regulation. The stimulator has been used in similar experiments across 
institutions, including the National Institute of Mental Health (NIMH), and has been approved by their IRB for 
use in humans.  
 
Psychosocial (non-medical) Risks 
Privacy: As per standard DCRP procedures, study data is recorded using standard forms.  All data will 
be stored in locked cabinets. For statistical analysis, only study IDs will be used as identifiers. Separate folders 
with unblinded information (e.g., patient name) will be kept in a locked cabinet in a separate office to ensure 
the blind.   
 
Safety Risk 
 
Though patients with severe suicidal ideation will be excluded from the study at screening, any patient 
who, based on the investigator’s judgment, is judged to present with an imminent risk of suicide will be 
discontinued from the study and referred to a local emergency room for further evaluation. 
 
VIII: Potential Benefits 
To Participating Individuals and Society 
 
The patients participating to the study may feel better, if for only a brief period of time. Some may not 
receive any direct benefit. The results of this study may lead to improved understanding of the neurobiology of 
depression, as well as the biomarkers for diagnosis and treating depression. Furthermore, the results from this 
study may lay the groundwork for future studies into the mechanism of action of ketamine’s antidepressant 
effects, as well as using neurobiology to subtype depression. 
 
IX: Monitoring and Quality Assurance 
 
Because this study is a physiological study of biomarkers, as opposed to a clinical trial, the principal 
investigator and co-investigators will be responsible for monitoring and quality assurance of the study. In 
conjunction with the research assistants, weekly meetings will be held and documented. Current participants in 
the study will be discussed at each meeting, and charts will be reviewed for completeness. 
 
Serious Adverse Events:  Expedited review will occur for all events meeting the FDA definition of SAEs 
– i.e., any fatal event, immediately life-threatening event, permanently or substantially disabling event, event 
requiring or prolonging inpatient hospitalization, or any congenital anomaly. This also includes any event that a 

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study investigator judges to impose a significant hazard, contraindication, side effect, or precaution.  Reporting 
to the IRB will be done within 24 hours of the SAE. 
 
Study Stopping Rules:  If at any time during the course of the study, the PI or CO-Is judge that risk to 
participants outweighs the potential benefits, the PI/CO-Is shall have the discretion and responsibility to 
recommend that the study be terminated.  
 
 
AE Reporting Guidelines 
Concomitant Medications and Adverse Events: Concomitant medications (dosage, start and stop dates) 
will be reviewed and recorded at each visit. The study doctor will document and side effect or adverse event 
during Phase II.  
In case of serious adverse cognitive side effects (delirium or confusion of clinical concern, with or 
without other symptoms like hallucinations, paranoia) or other serious side effects (e.g., cardiac, neurologic), 
patients will be discontinued from the study and immediately treated with the appropriate pharmacologic 
management (e.g., haloperidol, as needed, for delirium and psychotic symptoms). 
 
 
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