Understanding Effects of Cannabis Use and Abstinence on Neural Glutamate Homeostasis
Phase: EARLY_PHASE1
Conditions: Cannabis Use Disorder
Sponsor: Yale University

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CLINICAL STUDY PROTOCOL
Understanding effects of cannabis use and 
cessation on neural glutamate homeostasis
Principal Investigator
Stephen Baldassarri
Funded by
NIDA & Department Funds
Protocol Version
January 2024
Confidentiality Statement:
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NCT05664763

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2
Study Personnel
Principal Investigator
Stephen Baldassarri
Yale University
Email: stephen.baldassarri@yale.edu
Investigator
 Patrick  Skosnik
Yale University
Email: patrick.skosnik@yale.edu
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SYNOPSIS
Study Description 
PROJECT SUMMARY/ABSTRACT 
Cannabis use and availability continue to rise significantly in the US.  It is critical to expand 
our knowledge of the negative and positive effects of cannabis to "catch up" to the current 
reality of widespread and growing use. Cannabis and tetrahydrocannabinol (THC), its primary 
psychoactive chemical, have widespread effects on neural glutamate homeostasis, and 
specifically metabotropic glutamate receptor 5 (mGluR5).  mGluR5 regulates transmission of 
glutamate and plays a critical role in neural plasticity (i.e., long-term potentiation; LTP), 
memory, learning, mood, and addiction.  Specifically, it is thought that mGluR5 activation by 
glutamate initiates production of endocannabinoids (i.e., 2-AG) that bind retrograde to 
presynaptic cannabinoid receptor 1 (CB1).  This pathway inhibits further glutamate release 
and modulates synaptic plasticity diffusely in the brain.  However, cannabis binding to CB1 
disrupts this normal mechanism of glutamate homeostasis.  While the relationship between 
cannabis use and glutamate regulation has been explored in preclinical models, it has not been 
well-characterized in humans, and particularly in people with cannabis use disorder (CUD).  
Our preliminary data have demonstrated for the first time that mGluR5 availability in the 
fronto-limbic circuit, as measured by [18F]FPEB and positron emission tomography (PET), is 
higher in current cannabis users as compared with healthy controls (HC).  Our 
electroencephalography (EEG) data show that chronic exposure to cannabis is associated with 
disruptions in theta band (4-7 Hz) neural oscillations, which are important for memory 
processes, and dependent on intact CB1 and mGluR5 function.  Furthermore, a strong 
correlation linking impulsivity (an important clinical feature of CUD) with mGluR5 
availability was observed in people who use cannabis. We therefore propose to conduct the 
first in vivo human multimodal neuroimaging study exploring the relationship between 
mGluR5 availability (PET), neural oscillations (EEG), and cognitive function in people with 
CUD. The goal is to test the overall hypothesis that mGluR5 availability is higher in people 
with CUD compared with HC. This study will advance our understanding of cannabis effects 
on the neural glutamate system in humans and may lead to the development of novel 
therapeutics and biomarkers to treat people with CUD.  In Aim 1, we will determine 
differences in mGluR5 availability between people with CUD and HC in the fronto-limbic 
brain circuit. Aim 2 examines the associations between mGluR5 availability, CUD severity, 
neural oscillations, and cognitive function in CUD subjects. Aim 3 will determine how 
prolonged abstinence from chronic cannabis use affects mGluR5 availability, neural 
oscillations, and cognitive function in CUD subjects. The results from this innovative 
multimodal imaging study will improve our understanding of how chronic cannabis use and 
abstinence impacts neural glutamate receptors (mGluR5), and how this neurochemical 
mechanism relates to neural function (EEG) and clinically relevant functional measures of 
CUD (i.e., disease severity and impulsivity). 
 
 
 
 
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Objectives
SPECIFIC AIMS
Significance and Rationale: Cannabis is an addictive drug that is now widely available. 
Approximately 30% of cannabis users may have cannabis use disorder (CUD), and nearly 12 
million young adults used cannabis in 2019.5 Of note, there are currently no effective 
treatments for CUD.  There is an urgent need to better understand the consequences, and 
potential underlying causes, of CUD. Glutamate is the brain's primary excitatory 
neurotransmitter, and is strongly implicated in addictive disorders.6  Preclinical research 
indicates that chronic cannabinoid exposure disrupts glutamate homeostasis.7  However, the 
underlying mechanisms of this effect are incompletely understood in humans. Therefore, the 
goal of this proposal is to advance our mechanistic understanding of the consequences of 
CUD, with focus on metabotropic glutamate receptor 5 (mGluR5). This proposal has the 
potential to develop novel therapeutic targets for individuals with CUD.
mGluR5 in chronic cannabis use: mGluR5s are located mostly post-synaptically and on glia, 
and play a critical role in neural plasticity (i.e., long-term potentiation; LTP), memory, 
learning, mood, and addiction.8,9 Using [18F]FPEB and positron emission tomography (PET) 
neuroimaging, we can measure mGluR5 availability in vivo in people. Cannabis is known to 
significantly alter the same neurobehavioral functions mediated by mGluR5.7,10  Using 
electroencephalography (EEG), we have the capacity to relate mGluR5 availability with 
neural functions underlying memory and synaptic plasticity in people in vivo.
Preclinical models have revealed an intimate relationship between mGluR5 and the 
cannabinoid system.11  Specifically, mGluR5s on post-synaptic neurons regulate glutamate 
transmission through the synthesis of (natural) endocannabinoids (i.e. 2-AG). 2-AG binds to 
the presynaptic cannabinoid receptor 1 (CB1), which inhibits excessive glutamate release via 
negative feedback. Chronic cannabis use disrupts this homeostatic process, most notably 
leading to reduced synaptic glutamate release from pre-synaptic terminals via alterations in 
CB1 function.12-15 These pre-synaptic changes likely reflect compensatory mechanisms related 
to chronic cannabis exposure. However, no studies have examined the effect of chronic 
cannabis use on post-synaptic function in CUD. Given the central role of post-synaptic 
mGluR5 in the endocannabinoid system, it is critical to examine mGluR5 in people with 
CUD.   
Preliminary Data, Approach, and Aims: Our preliminary data demonstrate for the first time 
that mGluR5 availability in the fronto-limbic circuit (orbitofrontal, anterior cingulate, 
ventromedial prefrontal, and dorsolateral prefrontal cortices, hippocampus, and amygdala), as 
measured by [18F]FPEB PET neuroimaging, is higher in current cannabis users as compared 
with non-users (adjacent figure). In parallel, our preliminary EEG data show that chronic 
exposure to cannabis is associated with disruptions in theta band (4-7 Hz) neural oscillations, 
which are important for memory processes,16 and dependent on intact mGluR5 and CB1 
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function.17-21 Further, strong correlations linking neurocognition with mGluR5 availability 
were observed in cannabis users.  Thus, we propose to conduct the first ever in vivo human 
multimodal neuroimaging study (PET/EEG) examining the relationships between mGluR5 
availability, neural oscillations, and neurocognitive function in people with CUD.  Outcome 
measures are focused on clinically relevant markers of CUD severity.
Aim 1: To determine differences in fronto-limbic mGluR5 availability between people 
with cannabis use disorder (CUD; n = 30) and healthy controls (HC; n = 30).  Subjects 
will undergo [18F]FPEB PET neuroimaging to determine mGluR5 availability. Hypothesis 1: 
Fronto-limbic mGluR5 availability will be higher in individuals with CUD compared with 
HC. 
Aim 2: Within CUD subjects, to examine the associations between mGluR5 availability 
and: (a) CUD severity; (b) impulsivity; (c) EEG; and (d) neurocognition. CUD severity 
and impulsivity will be determined by validated instruments. EEG outcomes (e.g., theta 
power, LTP) known to be sensitive to both cannabis and glutamate will be collected during 
tasks of verbal memory22,23 and neural plasticity (long term potentiation; LTP).24  Hypothesis 
2: Higher mGluR5 availability will be associated with: (a) greater CUD severity (by DSM-5); 
(b) higher impulsivity; (c) lower theta-band power and LTP, and (d) worse performance on 
verbal memory.
Aim 3: Within CUD subjects, to determine changes in mGluR5 availability, EEG, and 
neurocognition following cannabis abstinence. CUD subjects from Aim 1 will repeat PET 
scanning, EEG, and cognitive testing following a 28-day period of confirmed abstinence aided 
by contingency management. Hypothesis 3: Following cannabis abstinence: (a) mGluR5 will 
decrease; (b) theta power/LTP will be increased; (c) memory will improve.
Impact: The results from this innovative multimodal imaging study will improve our 
understanding of how chronic cannabis use impacts glutamate homeostasis via measures of 
mGluR5, and how this neurochemical mechanism relates to clinically relevant measures of 
brain function (CUD severity, impulsivity, EEG, and cognitive function).  It will advance our 
mechanistic understanding of cannabis addiction and may lead to the development of novel 
therapeutics and biomarkers for people with CUD.
Endpoints
The primary endpoint is mGluR5 availability, as measured by FPEB, in cannabis-maintained 
and abstinent states.
Secondary endpoints are EEG outcomes and cognitive performance.
Study Population   
(1) Individuals with cannabis use disorder
(2) Healthy controls
Accrual Ceiling
Our target enrollment for study completion is 60 subjects: 30 cannabis use disorder (CUD) 
subjects and 30 healthy controls. 
Phase   
N/A
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Description of Sites/Facilities Enrolling Participants   
Yale PET Center, Yale MR Center
Description of Study Intervention
Research Design and Methods:
Research Subjects & Recruitment (Aims 1, 2, and 3)
Subjects: A total of 60 subjects will be recruited: 30 with cannabis use disorder (CUD) and 
30 healthy controls (HC) who do not use cannabis.  Each HC will be carefully matched to 
each CUD subject by age, sex, socioeconomic status, education, nicotine use, and other 
relevant variables.  CUD subjects will undergo PET, an optional EEG, and cognitive testing at 
Day 0 and Day 28.  HC will undergo these same measurements at Day 0 only. 
Some study participants will be also enrolled in HIC # 2000032181 (“SV2A Density Cannabis 
Use Disorder”), since that study has shared goals and study personnel with our study.  We 
have staff that are common to both studies.  If a subject is also enrolled in HIC# 2000032181, 
common procedures except for PET scanning will be performed once between the two studies 
(i.e., MRI and cannabis abstinence procedures will not be duplicated).  Whichever study the 
subject first signs up for will be where the abstinence data will be tracked.  The subject would 
sign a release of information to share the information with the other study.   
Recruitment and Screening: Subjects will be recruited with clinicians at Yale and in the 
greater Connecticut community with whom we have a long history of successful 
collaboration, including long-standing relations with the Yale Psychiatric Hospital inpatient 
and outpatient services and mental health clinics in the Greater New Haven area, as well as 
through flyers, public advertisement (newspaper, radio, internet postings), social media, and 
word of mouth.  Subjects will be appropriately compensated for their participation in the study 
and retained during the cannabis abstinence period using our validated contingency 
management paradigm.  
Study Schedule: Day 0: CUD subjects and healthy controls (HC) undergo PET scanning, an 
optional EEG, and cognitive testing. Days 1-27: CUD subjects abstain from cannabis use, 
aided by our validated contingency management strategy. Day 28: CUD subjects who are 
abstinent from cannabis use undergo repeat PET scanning, an optional EEG, and cognitive 
testing. Study Procedures: Eligible subjects will undergo an MRI scan for coregistration of 
PET data and for individualized source analysis of EEG. CUD subjects will be instructed not 
to use cannabis after midnight of Day 0, and last use of cannabis will be recorded. On Day 0, 
CUD subjects and HC will be scanned via PET, administered EEGs, and undergo cognitive 
testing. The same CUD subjects scanned on Day 0 will then be aided with cannabis abstinence 
and monitored over the next 4 weeks. On a weekly basis between Days 1-27, CUD subjects 
will be administered (1) the Withdrawal Discomfort score of the Cannabis Withdrawal 
Checklist, (2) the Cannabis Withdrawal Scale, which has subjective and objective (vital signs, 
weight) items, (3) Visual Analog Scale for Mood States, and (4) Pittsburgh Sleep Quality 
Index. CUD subjects will receive motivational enhancement and contingency management 
(CM) to promote and maintain abstinence. The escalating CM weekly payment schedule for 
cannabis abstinence is as follows: $50, $75, $125, $200, $650 (four-week abstinence bonus). 
If a participant is already getting paid to abstain from cannabis as part of their participation in 
another study (i.e. HIC# 2000032181), we will not pay them the usual amount ($1100) to 
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abstain for 28 days in our study. Instead, we will pay them a bonus of $400 which when 
combined with what they will get paid for abstaining for the other study will amount to more 
($400+1100) than what they would get if they abstained for just one study. If the subject is 
asked to come in CUD subjects will be required to come to the clinic two days a week for (1) 
motivational enhancement, (2) escalating contingency management, (3) verbal confirmation of 
abstinence, (4) urinary drug testing to detect cannabis exposure in the past 1-24 hours, (5) 
supervised collection of urine samples to assay THC-COOH, the principal metabolite of THC. 
To account for dilution, urinary THC-COOH:creatinine (T:C) ratio will be calculated. 
Abstinence will be confirmed if the weekly means of urinary T:C ratio decreases over time 
and does not increase by more than 50% relative to the prior specimen.[113-115] On Day 28, 
CUD subjects will undergo a second PET scan, an optional EEG, and cognitive testing. The 
CUD subjects who relapse between the first and second scans (expected to be 10% of CUD 
subjects) will be: 1) considered dropouts and 2) referred for treatment if so desired. We will 
replace dropouts with new CUD subjects to ensure maintenance of adequate statistical power 
for hypothesis testing. We have accounted for dropouts in the analytic plan, study timeline, 
and budget. Cannabis Assessments: CUD severity is assessed using DSM-5 criteria.[51] 
Cannabis withdrawal is assessed by the Cannabis Withdrawal Scale.[116] Cannabis craving is 
assessed using a 7-point scale. The Marijuana Problems Scale[117] will assess psychosocial 
impact of cannabis use. Multiple other clinically relevant features of CUD are described in the 
section below (Cognitive Testing and Behavioral Symptom Assessment). Detailed 
information on cannabis use patterns will be obtained using a time-line follow back approach: 
levels of cannabis consumption (estimated number of joints or edibles consumed) will be 
determined via interview for lifetime, the past six months, three months, one month, and then 
for the week prior to the test session as has been described previously [12,66,112,118,119]. 
Participants will be instructed to consider each day of the week and indicate, for an average 
week, how much they consumed per drug-use occasion for each length of time assessed. 
Cognitive Testing and Behavioral Symptom Assessment: The BIS/BAS will be used to assess 
trait impulsiveness and reward responsiveness.[120,121] Urgency, Premeditation (lack of), 
Perseverance (lack of), Sensation Seeking, Positive Urgency, Impulsive Behavior Scale will 
assess five impulsive personality traits. The Eriksen flanker task will supplement the 
assessment of the ability to suppress responses that are inappropriate to a particular 
context.[122] In addition, the Probabilistic Reward Task (PRT) will be used to provide an 
objective endophenotypic measure of reward responsiveness. On each of the scan days, CUD 
and controls subjects will also be administered neuropsychological tests from the CogState 
Cognitive Battery[123,124], which we previously showed to be sensitive to detecting 
cognitive deficits in mood disorders[125]: executive control (Groton Maze Learning task, 
GMLT; Set-Shifting task, SST and Go No-Go, GNG)[126-134] and working memory (One 
Back task, ONB, and Two Back task, TWB)[135-137]. Further, the battery has been well-
validated (studies using CogState batteries have been published in over 100 peer-reviewed 
journals). Most important for the current study, this battery is ideal for within-subject designs 
(i.e., it was specifically developed for repeated administration with minimal practice or 
learning effects). Finally, the CogState has been shown to have high test-retest reliability, 
thorough coverage of cognitive domains, comparable alternative forms, strong internal 
consistency, is well established in the general population, and has proven tolerability and 
acceptability. The entire battery takes approximately 30 minutes. For mood assessment, the 
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Hamilton Depression Rating Scale (HAM-D) and Montgomery-Asberg Depression Rating 
Scale (MADRS) will be administered by highly trained and reliable raters. These assessments 
will be administered at screening and at the time of PET scans.
C.1.d. Receptor Quantification Methods (Aim 1): (1) Magnetic Resonance Imaging 
Methods: MRI will be performed on a 3-Tesla Siemens Trio MR scanner (Siemens, Erlangen, 
Germany). A high resolution, 3-dimensional Magnetization Prepared Rapid Acquisition 
Gradient Echo (MPRAGE) T1-weighted sequence will be used to acquire sagittal images for 
anatomical determinations and co-registration (TR=1500ms, TE=2.83ms, FOV=256x256mm2, 
matrix=256x 256mm2, slice thickness=1.0mm without gap, 160 slices, voxel size 1.0 x 1.0 x 
1.0 mm3). The structural MRI data will serve to guide placement of ROIs for PET and for 
conducting GMM/partial volume correction (PVC) analyses.  We will acquire rs-fMRI and 
DTI at both imaging time points, and these will be examined on an exploratory basis.    A 
member of the research staff will accompany the study participant to the MR scans and stay 
for the duration of the session.  
(2) Positron Emission Tomography Methods: Data Acquisition: CUD subjects will 
participate in two [18F]FPEB scans, while controls will participate in one. Given we and others 
recently showed a diurnal variation in mGluR5 availability87,88, all mGluR5 scan collection 
will be at the same time of day. [18F]FPEB will be synthesized in the Radiochemistry 
Laboratory at the Yale PET Center.79 The HRRT, or a PET/CT, will be used to image 
subjects. [18F]FPEB will be administered as either a single bolus lasting up to one minute or 
bolus plus constant infusion lasting up to 2 hours. In either case, [18F]FPEB will be prepared 
in a single syringe and administered via software-controlled pump to achieve the desired 
radiotracer administration schedule not to exceed 5mCi. Syringe and lines will be assayed 
after scanning to correct for residual activity. Subjects are infused outside the scanner for 
the first 0-90 minutes during the pre-equilibrium period.  Then, they will be scanned for 
30 minutes in list-mode during the time the radiotracer reaches steady levels in the brain 
(90-120 minutes).79 At the beginning or end of each scan, a transmission scan, or low dose 
CT, will be obtained and used for attenuation correction. The emission list-mode data will 
then be binned into 5-min frames, and images will be reconstructed, correcting for attenuation, 
scatter, randoms, deadtime, and motion (using data from the Polaris Vicra using the MOLAR 
algorithm89). Pulse and blood pressure will be obtained before, during, and after scanning. 
Input Function Measurement: Venous concentrations of [18F]FPEB reach equilibrium by 60 
minutes post injection. Therefore, VT can be determined from the ratio of activity in tissue to 
that in venous blood samples. Blood samples will be acquired manually before and during the 
equilibrium period (90-120 minutes). If the individuals from CUD group are also participating 
in HIC# 2000032181 and already has an has an arterial line placed, blood may be drawn 
through the arterial line if intravenous access is unobtainable. Radioactivity concentration in 
whole blood and plasma will be measured using a gamma counter (Wizard 1480; Perkin-
Elmer, Waltham, MA, USA). Up to 6 tablespoons of blood will be drawn per PET scan. High-
performance liquid chromatography (HPLC) will be used to establish unmetabolized parent 
compound levels.79 The input function is calculated as the product of the interpolated parent 
fraction and the plasma concentration values. PET Image Analysis: To compute regional 
values, a summed PET image will be registered to each subject's T1- weighted MR image 
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using a six-parameter mutual information algorithm (FLIRT, FSL 3.2, Analysis Group, 
FMRIB, Oxford, UK), which in turn will be registered to an MR template image using 
nonrigid registration methods (Bioimage Suite, Yale University). Bioimage Suite atlas will be 
used to identify the primary regions of interest (ROIs) (Fronto-limbic circuit - hippocampus, 
amygdala, cingulate, dlPFC, vmPFC, and OFC). Outcome Measure Calculation: In humans, 
there is not a region that is completely devoid of mGluR581,90 — we and other have shown 
there is specific binding in the cerebellum.91,92 Therefore, we plan to examine VT.93 The VT 
images will be spatially normalized to a standard template (MNI, Montreal Neurological 
Institute) and analyses will be performed to assess ROI differences between groups ROIs. 
Gray Matter Masking: To partially account for partial volume effects, binary GMM will be 
employed. Individual MR images will be segmented with FAST (FMRIB's Automated 
Segmentation Tool, v3.1; The Analysis Group, FMRIB, Oxford, UK) to obtain gray matter 
(GM), white matter (WM), and cerebrospinal fluid (CSF) masks. The individual GMM images 
will be used to mask the candidate ROIs to obtain the mean regional values limited to GM 
voxels. GMM VT will be the primary outcome measure. Partial Volume Correction (PVC): We 
will also examine the effect of full partial volume correction on between-group differences in 
mGluR5 availability. PVC94,95 will be applied to the PET data on a frame-by-frame basis. The 
MR images will be segmented into GM, WM and CSF using FAST. Masked images will then 
be blurred by convolving the image with a Gaussian point spread function to achieve the 
resolution of the PET scanner (~3 mm). GM pixels will be corrected for spill-in and spill-out 
of activity from WM. Activity in CSF is assumed to be zero and activity in WM is assumed to 
be uniform and is estimated from each image.95 This method creates a set of PVC-corrected 
PET dynamic images, which are then used to create PVC VT images. Given the high resolution 
of the HRRT, we do not expect PVC to produce a substantial change in the data compared to 
the GMM analysis described above.
C.2.c Electroencephalography (EEG) Methods: 
General EEG Data Acquisition, Recording, and Preprocessing: EEG recording and 
preprocessing will be performed as described previously.21,101 Briefly, EEG data will be 
collected in an acoustically and electrically shielded booth, and recording will be done with 
the commercially available Active Two acquisition system (Biosemi, the Netherlands). A 
sampling rate of 1024 Hz will be utilized, with on-line low-pass filter of 256 Hz to prevent 
aliasing of high frequencies. A 64-channel electrode cap according to the extended 10-20 
system will be used, along with additional electrodes to record the vertical and horizontal 
electro-oculogram. All electrodes will be referenced during recording to a common-mode 
signal (CMS) electrode between POz and PO3 and then subsequently re-referenced to the nose 
offline. EEG data will be first bandpass filtered from 0.1 — 100 Hz (24 dB/oct) and notch 
filtered at 60 Hz.  Depending upon the particular stimulus parameters of each experiment (see 
below), the recorded EEG will be segmented into epochs consisting of the time period during 
stimulus presentation, along with a 100-300 ms baseline period. Any trial with a voltage 
greater than ± 100 µV will be considered an artifact (i.e., movement, etc.), and will be 
excluded from the analysis. Ocular movement correction will be applied using Gratton's 
algorithm.102 All EEG data preprocessing will be performed using the software package 
Analyzer 2.0 (Brain Products GmbH, Germany).  Time-Frequency Analyses: 
Induced/Evoked Power and Intertrial Coherence (Phase Locking Factor): Time-
frequency analyses will be the primary dependent measures in the proposed studies, along 
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with event-related potentials (ERPs), and will be performed as described previously.21,103 
Baseline normalized evoked/Induced power and Interelectrode Coherence (ITC; also termed 
phase locking factor) will be determined using a wavelet-based time-frequency spectrogram 
via Analyzer 2.0.  EEG Source Analysis: Standardized Low Resolution Brain 
Electromagnetic Tomography (sLORETA): Based on the scalp-recorded electric potential 
distribution, standardized low resolution brain electromagnetic tomography (sLORETA) will 
be used to compute the cortical three-dimensional distribution of current density. This will 
allow us to examine mGluR5 availability in ROIs in relation to EEG sources.  The particular 
form of standardization used in sLORETA endows the tomography with the property of exact 
localization to test point sources, yielding images of standardized current density with exact 
localization, albeit with low spatial resolution (i.e. neighboring neuronal sources will be 
highly correlated).104,105 sLORETA is the method of choice since it has no localization bias 
even in the presence of measurement and biological noise.104 Furthermore, sLORETA has 
been validated in several simultaneous EEG/fMRI studies,106,107 and in an EEG localization 
study for epilepsy.108 In the current implementation of sLORETA, computations will be made 
both in a realistic head model, 109 using the MNI152 template,110 and using each subject's 
structural MRI, with the three-dimensional solution space restricted to cortical gray matter, as 
determined by the probabilistic Talairach atlas.111 The intracerebral volume is partitioned in 
6239 voxels at 5 mm spatial resolution. C.2.d. EEG Paradigms:  RAVLT Task: A modified 
computer version of the Rey Auditory Verbal Learning Test (RAVLT) similar to Babiloni et 
al. (2009) will be utilized.22 The RAVLT will be administered while EEG data are collected in 
three separate conditions. In the passive listening condition, subjects will passively listen to a 
list of 15 words (presented one at a time) six times in a row. In the memory encoding 
condition, subjects will be administered a list of 15 words played one at a time, five times, and 
will be told to try and remember the list and to repeat as many words as possible after each 
list. A sixth distractor list will also be administered after the primary list as in the standard 
RAVLT. The inclusion of separate passive listening (which is not part of the standard 
RAVLT) and memory/encoding conditions will be included in order to compare neural 
signatures for both basic linguistic processing and processes related to memory encoding. 
Twenty minutes after the end of the encoding phase, subjects will be asked to repeat as many 
words as they can from the original list of words (the free recall phase). EEG will not be 
collected during the free recall phase. Following free recall, subjects will engage in a 
computerized recognition condition of the RAVLT. Subjects will hear words from the (1) 
original memorized list, (2) the distractor list, and (3) novel words not heard that day. Subjects 
will respond with a three-choice button box to indicate from which list each word originated. 
Words in the recognition condition will be presented every 4 seconds. This EEG version of 
the RAVLT allows separate examinations of neural oscillations during passive listening, 
memory encoding, and recognition. In addition, the words will be post-hoc sorted based on 
correct performance to examine activity during encoding for words that were accurately 
recalled (the "subsequent memory effect").  LTP Task: We induce and measure LTP using 
EEG with repetitive sensory stimulation.100  This paradigm has been shown to have the 
properties of LTP including (1) frequency dependency, (2) temporal stability, (3) input 
specificity, and (4) de-potentiation by low frequency stimulation.100 Tone pips (tone frequency 
1000 Hz, presented for 50 msec) will be used in all stages of the task (i.e., for baseline, 
tetanus, and post-tetanic recording). During the baseline recording, 120 tone pips will be 
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presented with a variable inter-stimulus interval between 1800 — 2600 msec. After the 
baseline recording, LTP will be induced using auditory tetanus. The tetanus will consist of 
two minutes of the tone pips presented at 13 Hz. Subsequently, the test phase will commence, 
which will consist of another presentation of 120 tone pips as in the baseline period. The 
primary outcome measure will be N100-P200 amplitude after the auditory tetanus versus 
before the tetanus. The N100-P200 complex will be determined via automated algorithms and 
verified manually as described previously.112
Study Duration
5 years
Participant Duration   
1 month
Schema
Schedule of Activities (SoA)
OUTCOMES
SOURCE/MEASURE HC
CUD 
(baseline)
Day 0
CUD (abstinence)
Day 28
Neural Receptors
 
mGluR5 
Availability 
PET ROI's VT: OFC, 
anterior cingulate, 
vmPFC, dlPFC, 
hippocampus, 
amygdala
X
X
X
Cannabis Use Disorder — Clinically Relevant Functional 
Measurements
 
CUD Severity
DSM-5
 
X
 
Cannabis Use 
Patterns
Timeline Follow Back
 
X
X
Cannabis 
Withdrawal 
Severity
Cannabis Withdrawal 
Scale
 
X
X
Cannabis 
Craving
7-point Scale
 
X
X
Cannabis-
Related 
Problems
Marijuana Problems 
Scale
 
X
 
Impulsivity
BIS/BAS
X
X
 
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Reward 
Processing
Probabilistic Reward 
Task
X
X
X
Mood
HAM-D, MADRS, 
Visual Analog Scale 
for Mood States
X
X
X
Sleep
Pittsburgh Sleep 
Quality Index
X
X
X
Cannabis Biomarkers
 
Cannabis Use 
Status
Urinary THC-COOH*
X
X
X
Neural 
Function
 
 
 
 
Verbal Memory EEG: Theta band 
power/coherence
X
X
X
Synaptic 
Plasticity / LTP
EEG: N100-P200 
amplitude increase 
after auditory tetanus
X
X
X
Neurocognitive 
Function
CogState Cognitive 
Battery
X
X
X
End of Study Definition
A participant is considered to have completed the study if he or she has completed all phases 
of the study including the last visit or the last scheduled procedure shown in the Schedule of 
Activities (SoA), Section 1.3.
The end of the study is defined as completion of the last visit or procedure shown in the SoA 
in the trial globally.
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Table of Contents
SYNOPSIS..................................................................................................................................3
1 - Statement of Compliance.....................................................................................................15
1.1 Statement of Compliance.................................................................................................15
2 - Introduction .........................................................................................................................16
2.1 Study Rationale................................................................................................................16
2.2 Background......................................................................................................................16
2.3 Risk/Benefit Assessment.................................................................................................20
2.3.1 Known Potential Risks..............................................................................................20
2.3.2 Known Potential Benefits..........................................................................................22
2.3.3 Assessment of Potential Risks and Benefits .............................................................22
3 - Objectives and Endpoints....................................................................................................25
3.1 Objectives and Endpoints................................................................................................25
4 - Study Design........................................................................................................................26
4.1 Overall Design.................................................................................................................26
4.2 Scientific Rationale for Study Design .............................................................................26
4.3 Justification for Dose.......................................................................................................30
4.4 End of Study Definition...................................................................................................30
5 - Study Population..................................................................................................................32
5.1 Inclusion Criteria.............................................................................................................32
5.2 Exclusion Criteria............................................................................................................32
5.3 Lifestyle Considerations..................................................................................................32
5.4 Screen Failures ................................................................................................................33
5.5 Strategies for Recruitment and Retention........................................................................33
6 - Study Intervention ...............................................................................................................35
6.1 Study Intervention(s) Administration..............................................................................35
6.1.1 Study Intervention Description .................................................................................35
6.1.2 Dosing and Administration .......................................................................................36
6.2 Preparation/Handling/Storage/Accountability.................................................................36
6.2.1 Acquisition and accountability..................................................................................36
6.2.2 Formulation, Appearance, Packaging, and Labeling ................................................36
6.2.3 Product Storage and Stability....................................................................................36
6.2.4 Preparation ................................................................................................................36
6.3 Measures to Minimize Bias: Randomization and Blinding.............................................36
6.4 Study Intervention Compliance.......................................................................................36
6.5 Concomitant Therapy ......................................................................................................37
6.5.1 Rescue Medicine .......................................................................................................37
7 - Study Intervention Discontinuation and Participant Discontinuation/Withdrawal .............38
7.1 Discontinuation of Study Intervention ............................................................................38
7.2 Participant Discontinuation/Withdrawal from the Study ................................................38
7.3 Lost to Follow-Up ...........................................................................................................38
8 - Study Assessments and Procedures.....................................................................................39
8.1 Efficacy Assessments ......................................................................................................39
8.2 Safety and Other Assessments.........................................................................................40
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8.3 Adverse Events and Serious Adverse Events..................................................................41
8.3.1 Definition of Adverse Events (AE)...........................................................................41
8.3.2 Definition of Serious Adverse Events (SAE)............................................................41
8.3.3 Classification of an Adverse Event ...........................................................................41
8.3.3.1 Severity of Event....................................................................................................42
8.3.3.2 Relationship to Study Intervention.........................................................................42
8.3.3.3 Expectedness ..........................................................................................................43
8.3.4 Time Period and Frequency for Event Assessment and Follow-Up .........................43
8.3.5 Adverse Event Reporting ..........................................................................................43
8.3.6 Serious Adverse Event Reporting .............................................................................44
8.3.7 Reporting Events to Participants...............................................................................44
8.3.8 Events of Special Interest..........................................................................................44
8.3.9 Reporting of Pregnancy (if applicable) .....................................................................44
8.4 Unanticipated Problems...................................................................................................44
8.4.1 Definition of Unanticipated Problems (UP)..............................................................44
8.4.2 Unanticipated Problem Reporting.............................................................................45
8.4.3 Reporting Unanticipated Problems to Participants ...................................................46
9 - Statistical Considerations ....................................................................................................47
9.1 Statistical Hypotheses......................................................................................................47
9.2 Sample Size Considerations ............................................................................................47
9.3 Populations for Analyses.................................................................................................49
9.4 Statistical Analyses..........................................................................................................49
9.4.1 General Approach  ...................................................................................................49
9.4.2 Analysis of the Primary Efficacy Endpoint(s) ..........................................................49
9.4.3 Analysis of the Secondary Endpoint(s).....................................................................49
9.4.4 Safety Analyses ............................................................................................................50
9.4.5 Baseline Descriptive Analyses (if applicable)..............................................................50
9.4.6 Planned Interim Analyses (if applicable) .....................................................................50
9.4.7 Sub-Group Analyses.....................................................................................................50
9.4.8 Tabulation of Individual Participant Data ....................................................................50
9.4.9 Exploratory Analyses ...................................................................................................50
10 - Supporting Documentation and Operational Considerations ............................................51
10.1 Regulatory, Ethical, and Study Oversight Considerations ............................................51
10.1.1 Informed Consent Process  .....................................................................................51
10.1.1.1 Consent/Assent and Other Informational Documents Provided to Participants  51
10.1.1.2 Consent Procedures and Documentation  ............................................................51
10.1.2 Study Discontinuation and Closure  .......................................................................51
10.1.3 Data Confidentiality and Participant Policy  ..........................................................52
10.1.4 Future Use of Stored Human Specimens and Data  ...............................................52
10.1.5 Safety Oversight  ....................................................................................................53
10.1.6 Key Roles and Study Governance..............................................................................53
10.1.7 Clinical Monitoring ....................................................................................................53
10.1.8 Quality Assurance and Quality Control  ................................................................53
10.1.9 Data Handling and Record Keeping...........................................................................53
10.1.9.1 Data Collection and Management Responsibilities  ...........................................53
10.1.9.2 Study Records Retention  ....................................................................................54
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10.1.10 Protocol Deviations  .............................................................................................54
10.1.11 Publication and Data Sharing Policy  ...................................................................54
10.1.12 Conflict of Interest Policy .....................................................................................55
10.2 Additional Considerations.............................................................................................55
10.3 Protocol Amendment History........................................................................................55
10.4 Abbreviations.................................................................................................................55
LIST OF TABLES....................................................................................................................56
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1 - Statement of Compliance
1.1 Statement of Compliance
The trial will be carried out in accordance with International Conference on Harmonization Good 
Clinical Practice (ICH GCP) and the following: 
o
United States (US) Code of Federal Regulations (CFR) applicable to clinical 
studies (45 CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, 
and/or 21 CFR Part 812) 
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are 
responsible for the conduct, management, or oversight of NIH-funded clinical trials have 
completed Human Subjects Protection and ICH GCP Training.
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the Institutional Review Board (IRB) for review and approval.  Approval of both 
the protocol and the consent form must be obtained before any participant is enrolled.  Any 
amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study.  In addition, all changes to the consent form will be IRB-approved; a 
determination will be made regarding whether a new consent needs to be obtained from 
participants who provided consent, using a previously approved consent form.
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2 - Introduction
2.1 Study Rationale
Significance and Rationale: Cannabis is an addictive drug that is now widely available. 
Approximately 30% of cannabis users may have cannabis use disorder (CUD), and nearly 12 
million young adults used cannabis in 2019.5 Of note, there are currently no effective treatments 
for CUD.  There is an urgent need to better understand the consequences, and potential 
underlying causes, of CUD. Glutamate is the brain's primary excitatory neurotransmitter, and is 
strongly implicated in addictive disorders.6  Preclinical research indicates that chronic 
cannabinoid exposure disrupts glutamate homeostasis.7  However, the underlying mechanisms of 
this effect are incompletely understood in humans. Therefore, the goal of this proposal is to 
advance our mechanistic understanding of the consequences of CUD, with focus on 
metabotropic glutamate receptor 5 (mGluR5). This proposal has the potential to develop 
novel therapeutic targets for individuals with CUD.
2.2 Background
1. A.1. Overview: How does cannabis affect glutamate receptors in the human brain?  
Cannabis has a wide range of health and behavioral effects, many of which remain 
incompletely understood.10 Yet, both recreational and medicinal use of the drug are 
widespread and continuing to increase in the US.  Cannabis is addictive. Nearly 30% of 
users may have cannabis use disorder (CUD), and nearly 12 million young adults used 
cannabis in 2019.5 Recreational use is now legal in 15 states, and medical use is legal in 
35 states. As more states continue to legalize use for recreational purposes, cannabis use 
will likely continue to increase.25 This could result in an increasing number of people 
experiencing negative effects, including addiction.25Cannabis use impairs cognition, 
memory, and motor coordination.  With more prolonged or heavy use, it increases the 
risk of developing schizophrenia or other psychoses.26  On the other hand, it is 
hypothesized that cannabis may have therapeutic effects in numerous neurologic 
conditions such as chronic pain, neurodegenerative diseases, and some psychiatric 
conditions (i.e. PTSD, anxiety, and sleep disorders). It is critical to more fully understand 
the neural mechanisms of cannabis to minimize harmful consequences and maximize 
therapeutic benefit. Tetrahydrocannabinol (THC) is the primary psychoactive component 
of cannabis.  It is well-established that many of the psychoactive and addictive properties 
of cannabis are mediated by the neural circuit involving THC binding to the cannabinoid 
receptor 1 (CB1), a presynaptic receptor that is widely distributed throughout the central 
nervous system.  Less well-understood, however, is the impact of CB1-THC binding on 
postsynaptic receptors such as metabotropic glutamate receptor 5 (mGluR5), a key 
regulator of glutamate homeostasis and endocannabinoid synthesis.11  There is strong 
evidence from the preclinical literature that cannabinoid exposure alters glutamate 
homeostasis via mGluR5 and CB1, which may mediate many important neural effects. 
Indeed, there is a close relationship between the cannabinoid and glutamatergic 
systems.19 However, this relationship has not been adequately studied in humans, and the 
degree to which disturbances in the glutamate system influences the potential harms and 
therapeutic benefits from cannabis use is unclear.  It is critical to advance our 
understanding of how chronic cannabis exposure affects the brain, including the 
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consequences of these changes on neural function.  Importantly, we need to better 
understand whether neural dysregulation normalizes when cannabis use stops. This 
innovative multimodal (PET/EEG/neurocognitive) study will expand our knowledge and 
thus advance our understanding of the neurobehavioral mechanisms underlying cannabis 
addiction, with a focus on the metabotropic glutamate receptor 5 (mGluR5), in human 
subjects.    A.2. Metabotropic Glutamate Receptor 5 (mGluR5): Importance in 
addiction and related behavioral phenotypes: Glutamate is the brain's primary 
excitatory neurotransmitter, and is critical for optimal neuronal function.27 Glutamate 
transmission between neural circuits linking key limbic and cortical brain regions 
maintains a balance between the reward-seeking behaviors necessary for survival and 
higher executive functions.28mGluR5, a key regulator of glutamate homeostasis, is a G 
protein-coupled receptor located mostly on post-synaptic neurons diffusely throughout 
the brain, especially key areas involved in addiction such as hippocampus, amygdala, 
striatum, prefrontal cortex, and cingulate.29-31  It regulates synaptic plasticity of 
glutamatergic neurons, which is fundamental to learning and neural transmission of 
critical information.  Alterations in mGluR5 receptor availability have been implicated in 
addiction32 and its related endophenotypes such as executive function impairment,33 
mood disorders,34 and impulsivity.35  Preclinical studies of mGluR5 knockout mice 
and/or the use of mGluR5 antagonists have revealed several key findings pointing to the 
likely involvement of mGluR5 in addictive disorders including: (1) reduced drug self-
administration and reduced drug-seeking behavior;3637 (2) attenuation of drug-induced 
rewarding effects38 and conditioned place preference39; and (3) reduced withdrawal.40,41 
The precise mechanisms whereby these processes occur remain unclear. These data, 
combined with observations of mGluR5 effects on drugs of abuse in general,42,43 make 
mGluR5 a potentially important molecular target in individuals with cannabis use 
disorder (CUD).A.3. mGluR5: Importance in synaptic plasticity: Synaptic plasticity 
is critical for neuronal adaptations to a wide range of environmental conditions.  Local 
changes in synaptic strength [i.e., long-term potentiation (LTP) or depression (LTD)] 
underlie much of the brain's ability to change its physical structure in response to changes 
in the environment, particularly during learning and memory.  mGluR5 regulates 
synaptic plasticity of glutamatergic neurons, which is fundamental to learning and 
neural transmission of critical information.44,45  Activation of mGluR5 induces LTD in 
hippocampal area CA1 and cerebellar Purkinje neurons.46,47  Other preclinical studies 
have shown that antagonism of mGluR5 impaired LTP, spatial memory, and suppressed 
theta (5-10 Hz) activity in the dentate gyrus.17,18  Interestingly, the cannabinoid system is 
also implicated in learning, memory, and synaptic plasticity, indicating a potential 
convergence zone wherein mGluR5 and CB1 may interact functionally.48 Given the 
overlap between cannabis use, synaptic plasticity, learning, and cognition, it is critical to 
study mGluR5 in the context of cannabis use. 
a.
SIGNIFICANCE 
Figure 1: mGluR5-CB1 relationship.  mGluR5 is a key regulator of glutamate homeostasis 
via cannabinoid receptor 1 (CB1).  The consumption of exogenous CB1 ligands via cannabis 
use may profoundly alter this process.  The focus of this grant is on how chronic cannabis use 
and abstinence specifically affects mGluR5 availability in humans with cannabis use disorder.  
Figure Source: Cristino, et.al., 2019 (See ref 11).
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A.4. mGluR5: Key regulator of normal glutamate homeostasis via endocannabinoid 
synthesis: Under normal physiological conditions, preclinical models suggest that mGluR5 is a 
key regulator of glutamate homeostasis via CB1 at both excitatory (glutamatergic) and 
inhibitory (GABAergic) synapses, as shown in Figure 1 and described here.11  The focus of this 
grant is on understanding how chronic cannabis use and abstinence affect mGluR5 availability in 
humans with CUD. MGluR5 regulates glutamate primarily by triggering endocannabinoid (ECB) 
production.  ECBs (e.g., 2-AG) are naturally occurring molecules that regulate neurotransmitter 
release and synaptic plasticity by binding to presynaptic CB1. Under normal physiological 
conditions, stimulation of presynaptic excitatory neurons leads to glutamate release into the 
synaptic cleft and peri-synaptically.49 Glutamate binding to post-synaptic mGluR5 triggers the 
"on-demand" synthesis of the endocannabinoid 2-AG.  2-AG then binds in a retrograde manner 
to CB1, leading to synaptic depression and inhibition of further glutamate release.  Thus, under 
normal conditions, glutamate homeostasis is maintained via a negative feedback loop 
mediated by mGluR5 and endocannabinoid binding to presynaptic CB1.  This process may 
be disrupted by the consumption of exogenous cannabinoids.50
Thus, mGluR5 is a critical, widespread molecular target involved in neural glutamate 
homeostasis that interacts directly with the cannabinoid system and is significantly involved in 
neural plasticity, memory, addiction, executive function, cognition, and emotional processing.  It 
is therefore critically important to study the relationship between CUD and mGluR5, and to 
relate this to neural function and behavioral outcomes related to addiction.  
A.5. Cannabis Use Disorder (CUD): Disruption of glutamate homeostasis via mGluR5 and 
CB1 (Aim 1): CUD is characterized clinically as a syndrome involving impaired control over 
cannabis use, problems occurring as a result of cannabis use, and signs of physiologic 
dependence (i.e. tolerance or withdrawal upon abstinence).51 Mechanistically, CUD is mediated 
primarily by recurrent and chronic THC binding to CB1.52  Cannabis and THC have widespread 
effects on neural glutamate transmission.50  Specifically, preclinical models have demonstrated 
that THC binding to CB1 inhibits glutamate release and leads to long-term synaptic depression 
of glutamatergic neurotransmission.53,54 Animal studies have demonstrated that after chronic 
exposure to THC and other CB1 agonists, there is a reduction in the number and function of CB1 
receptors.55-60 Furthermore, CB1 downregulation and desensitization has a distinct regional and 
temporal course, and is related to the duration and magnitude of exposure, with greater 
downregulation in cortical compared with subcortical regions.55,61,62 With prolonged abstinence, 
there is normalization in the number and function of presynaptic CB1 over 2 weeks, with faster 
recovery in subcortical compared to cortical regions.59  However, it is unknown what happens 
to postsynaptic mGluR5 from chronic cannabis use and following cannabis abstinence.  We 
plan to fill this knowledge gap through the proposed innovative and rigorous study.  As we 
illustrate in our robust preliminary data below (Sections C.1a., C.2.a., and C.3.a.), our central 
hypothesis is that mGluR5 upregulation occurs as a result of chronic cannabis use and may 
reverse upon cannabis abstinence.
A.6. Imaging cannabis use in humans (Aim 1): Imaging studies in humans have demonstrated 
two key findings regarding chronic cannabis use: (1) lower neural glutamate release14,15 and (2) 
CB1 downregulation,63,64 which has been shown to normalize after 4 weeks of abstinence.  These 
findings suggest changes in both CB1 number and function from chronic cannabis use.  In the 
first in vivo study of CB1 in cannabis users, Hirvonen et al., using positron emission tomography 
(PET) imaging and [18F]FMPEP-d2, demonstrated that chronic, heavy cannabis users showed 
~20% lower CB1 availability relative to controls.63 Consistent with animal studies, this reduction 
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occurred in cortical but not in subcortical brain regions. In addition to showing lower CB1 
availability among people with chronic cannabis use, emerging data suggest that with abstinence 
of cannabis use, there is some recovery of CB1 receptor availability. We found that whereas 
there was a statistically significant reduction (~-15%) in CB1 availability in cannabis users at 
baseline relative to controls, these deficits persisted but were no longer statistically significant 
after 2 and 28 days of abstinence.  This suggests that there is some recovery of CB1 availability 
over time following cannabis abstinence.12 Collectively, these findings were interpreted as 
evidence of CB1 downregulation with chronic cannabis use that reversed with abstinence.
            A more recent study found lower mGluR5 availability in youth (ages 18-20 years) who 
were at-risk for substance use disorders (i.e., based on high impulsivity and aggression), 
including cannabis.65  No main effects of cannabis use alone on mGluR5 were observed.  
However, the study was limited by small subgroups (i.e., n = 3 CUD subjects) and uncertain 
timing and pattern of cannabis use relative to PET scanning.  Furthermore, the cross-sectional 
nature of the study limited the ability to understand dynamic changes in mGluR5 over time based 
on cannabis use status.  Our proposed study design, with scans performed longitudinally during 
cannabis-maintained and cannabis-abstinent states in the same subjects, will help more 
definitively determine the relationship between chronic cannabis use and mGluR5.  
A.7. Central Hypothesis: Chronic Cannabis Use Increases mGluR5 Availability (Aim 1): 
The observation that chronic cannabis use leads to both lower synaptic glutamate release and 
CB1 downregulation raises a critical question: How do cannabis-induced changes in CB1 and 
glutamate release alter glutamate homeostasis?  We propose it occurs via increased mGluR5 
availability.  Normally, CB1 on presynaptic glutamatergic terminals are activated by retrograde 
endocannabinoids (i.e., 2-AG) that reduce Ca2+ influx, thus playing a modulatory role by 
inhibiting glutamate release (Figure 2, Left). While chronic cannabis exposure decreases the 
number of CB1 receptors (downregulation), constant heavy use of cannabis leads to tonically 
activated CB1 via THC (despite the known downregulation) (Figure 2, Right). This would 
result in decreased and unmodulated firing in glutamatergic pyramidal cell networks, thus 
leading to disruptions in functionally relevant neural activity (i.e., theta oscillations).19  We 
therefore propose that chronic cannabis use leads to less pre-synaptic glutamate release, less 
glutamate binding to mGluR5, and hence compensatory upregulation of mGluR5 (i.e., higher 
mGluR5 availability). Thus, it is clear that chronic exposure to THC from cannabis likely leads 
to profound disruption of glutamate homeostasis.  PET is the best imaging modality for 
studying changes in neuroreceptors in vivo and is the focus of Aim 1 of this proposal.  Using 
[18F]FPEB PET neuroimaging, we will measure mGluR5 availability in vivo in  people with 
cannabis use disorder (CUD) and compare with healthy controls. 
A.8. Cannabis Use Disorder: Insights from Electroencephalography (EEG) (Aim 2): While 
PET is the best imaging modality for studying changes in neuroreceptors in-vivo, this method is 
unable to provide information regarding how receptor changes affect neural function. By 
contrast, EEG is one of the few available neuroimaging methodologies that can directly 
measure neural events (postsynaptic potentials) with high temporal precision in humans. Thus, 
combining PET and EEG can be a powerful tool with which to examine the relationship 
between receptor dynamics and neural function.  EEG effects from chronic cannabis use, along 
with validated cognitive assessments, provide highly relevant clinical correlates to mGluR5 
availability.
The fact that CUD has disruptive effects across broad domains of brain function suggests a 
common neurophysiological mechanism. One potential mechanism may involve the role of CB1 
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in modulating glutamate function, and hence the inhibitory-excitatory (I-E) balance of neural 
circuits. Skosnik et al. (2006) hypothesized that the neurobehavioral effects of cannabis use may 
involve alterations in neural synchronization.66 The impetus for this idea stems from the fact 
that interactions between glutamatergic pyramidal cells and GABAergic interneurons are 
involved in generating and maintaining neural oscillations in the theta and gamma (31-80 Hz) 
range.19,67-73 This may be particularly germane to the effects of cannabis, as synchronized neural 
oscillations are thought to be involved in memory and learning (domains of function that are 
known to be disrupted by cannabinoid agonists).  The effect of altered I-E balance may be 
particularly relevant to theta oscillations, as theta has been shown to be mediated in part by both 
CB1 and mGluR5 (as discussed above). From a functional standpoint, it has been established that 
theta may represent a "neural code" of learning and memory, and is related to synaptic 
plasticity.16,74,75  The relationships between mGluR5 availability, CUD severity, neural 
oscillations, and other clinically relevant domains (i.e., impulsivity, cognitive function) in people 
with CUD are the focus of Aim 2.  Using EEG, we now have the capacity to measure neural 
function underlying memory and synaptic plasticity in human subjects in-vivo through 
theta band (4-7 Hz) neural oscillations and tetanic sensory stimulation, respectively.
A.9. Cannabis Use Disorder: Abstinence and the need for treatments (Aim 3): There is a 
substantial amount of misinformation regarding the addictive potential of cannabis (particularly 
on social media), leading to a general underappreciation of the issue among the public.76  
Estimates suggest that nearly 30% of cannabis users may have cannabis use disorder, and 10% 
will develop addiction (severe use disorder).7  Aim 3 focuses on the neural effects of prolonged 
(i.e., 4 weeks) cannabis abstinence in people with CUD.  It is critical to study CUD in human 
subjects longitudinally to advance our mechanistic understanding of disease pathogenesis and 
identify novel treatment targets.  Studying mGluR5, cognitive function, and EEG at two distinct 
timepoints in the addiction process (cannabis use and abstinence) will allow us to determine the 
long-lasting neurologic changes that might perpetuate CUD and influence disease severity, as 
well as changes occurring as a result of prolonged cannabis abstinence.
Lastly, it is worth noting that there are no current FDA-approved treatments for cannabis use 
disorder, despite the availability of several cannabinoid receptor agonists.  Aside from the 
known changes in CB1 number and function, the mechanisms underlying cannabis use 
disorder are still not fully understood and need further study.  Alterations in glutamate 
homeostasis are a likely contributing disease factor, given glutamate's central nature in 
endocannabinoid signaling.  mGluR5 likely plays an important role in this process.  
Developing our understanding of effects on glutamate homeostasis resulting from chronic use of 
cannabis and subsequent abstinence from the drug will advance our ability to develop new 
treatment targets and therapeutics for this population.  Collectively, these issues underscore the 
urgent need to understand the effects of chronic cannabis exposure on the brain, and importantly, 
whether these effects reverse when cannabis use stops.
A.10. Summary: Significance of the current proposal: Research efforts have been unable to 
keep pace with rapidly increased public demand for cannabis products, progressive legalization, 
and widespread use.  The preclinical literature provides a biologic basis for studying the effects 
of chronic cannabis use on the neural glutamate system.  However, it is critical to advance our 
understanding of cannabis use effects in human subjects.  Our preliminary data, described in 
further detail below (Sections C.1.a., C.2.a., and C.3.a.), indicate for the first time that mGluR5 
availability is significantly higher in cannabis users as compared with healthy controls. 
Among subjects who use cannabis chronically, mGluR5 availability may normalize following a 
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period of cannabis abstinence.  Neural oscillations link directly with glutamatergic 
transmission and are disrupted from chronic cannabis use.  Finally, mGluR5 availability is 
strongly correlated with psychiatric symptoms and may predict disease severity in this 
population, indicating that mGluR5 is a clinically relevant biomarker of mental health in 
people with cannabis use disorder.  It is therefore both timely and innovative to study the effects 
of cannabis use on mGluR5 in humans with CUD using multimodal imaging (PET/EEG).  This 
study has the potential to increase our understanding of the mechanisms by which cannabis use 
affects the brain and is addictive.  It may lead to novel treatment targets among people who 
suffer from CUD.
 
2.3 Risk/Benefit Assessment
2.3.1 Known Potential Risks
Potential risks: Potential risks and discomforts from this study include 1) risks associated with 
venous catheter insertion and with blood drawing, 2) risks associated with radiation exposure, 3) 
risks associated with MR, 4) risks associated with cannabis withdrawal.
 
1. Risks Associated with Blood Drawing and IV line Insertion: Drawing blood and 
inserting an intravenous line (IV) into an arm vein are safe and standard medical 
procedures. Sometimes a bruise will occur at the puncture site and rarely a blood clot or 
infection will occur in the vein. Certain individuals may feel light-headed during 
venipuncture. The volume of blood collected during this study, include screening 
laboratories, MRI and PET scans, will be approximately 16 tablespoons. The combined 
amount of blood between the two study visits on a given study day will not exceed 100 
mL, and the timeframe between each visit day is over 2 weeks apart. This is not expected 
to have any serious negative effects on a study participant.
2. Risks Associated with Radiation: The Yale University Radioactive Investigational 
Drug Committee (RIDC) and Radiation Safety Committee (RSC) have reviewed and 
approved the use of radiation in this research study. This research study involves 
exposure to radiation from [18F]FPEB PET scanning. This radiation exposure is not 
necessary for medical care and is for research purposes only. 
The targeted amount of radiation an individual subject can receive in this study is from 
up to 2 injections (HC) with a total of ≤10 mCi (up to 5mCi per injection) or up to 3 
injections (CUD) with a total of ≤15 mCi (up to 5mCi per injection) from [18F]FPEB, 
plus transmission scans, or low dose CT scans of the brain. The maximum amount of 
radiation exposure subjects will receive in this study is equal to an effective dose of .714 
rem (HC) or 1.071 rem (CUD).
The amount of radiation involved in this research is small, but may slightly increase the 
risk of getting cancer. Scientists are not certain about the actual cancer risk at these low 
doses, and there may be no risk at all, but to be conservative we assume that any amount 
of radiation may pose some increased cancer risk.
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3. Risks associated with magnetic resonance imaging: Magnetic resonance 
(MR) is a technique that uses magnetism and radio waves, not x-rays, to take 
pictures and measure chemicals of various parts of the body. The United States 
Food and Drug Administration (FDA) has set guidelines for magnet strength and 
exposure to radio waves, and we carefully observe those guidelines.
 
Subjects will be watched closely throughout the MR study.  Some people may feel 
uncomfortable or anxious.  If this happens, the subject may ask to stop the study at any time and 
we will take them out of the MR scanner.  On rare occasions, some people might feel dizzy, get 
an upset stomach, have a metallic taste or feel tingling sensations or muscle twitches. These 
sensations usually go away quickly and research staff will monitor for them.
 
There are some risks with an MR study for certain people. If a subject has a pacemaker or some 
metal objects inside their body, they may not be in this study because the strong magnets in the 
MR scanner might harm them.  Another risk is the possibility of metal objects being pulled into 
the magnet and hitting the subject.  To reduce this risk we require that all people involved with 
the study remove all metal from their clothing and all metal objects from their pockets. We also 
ask all people involved with the study to walk through a detector designed to detect metal 
objects.  It is important to know that no metal can be brought into the magnet room at any time.  
Also, once subjects are in the magnet, the door to the room will be closed so that no one from 
outside accidentally goes near the magnet.
 
This MR study is for research purposes only and is not in any way a clinical examination. The 
scans performed in this study are not designed to find abnormalities. The primary investigator, 
the lab, the MR technologist, and the Magnetic Resonance Research Center are not qualified to 
interpret the MR scans and are not responsible for providing a diagnostic evaluation of the 
images.  If a worrisome finding is seen on a subject's scan, a radiologist or another physician will 
be asked to review the relevant images.  Based on his or her recommendation (if any), the 
primary investigator or consulting physician will contact the subject, inform them of the finding, 
and recommend that they seek medical advice as a precautionary measure. The decision for 
additional examination or treatment would lie solely with the subject and their physician. The 
investigators, the consulting physician, the Magnetic Resonance Research Center, and Yale 
University are not responsible for any examination or treatment that subjects receive based on 
these findings. The images collected in this study are not a clinical MR exam and for that reason, 
they will not be made available for diagnostic purposes.
 
1. Cannabis Abstinence / Withdrawal: 
 
Cannabis withdrawal: There is a risk that cannabis-dependent subjects will experience 
discomfort related to withdrawal from cannabis. The typical withdrawal symptoms include 
irritability, anxiety, anger, aggression, appetite change, weight loss, restlessness, altered sleep, 
strange dreams and physical discomfort.24,25  Less common symptoms include chills, depressed 
mood, stomach pain, and sweating. The severity of cannabis withdrawal has been equated with 
the level of withdrawal from tobacco and caffeine. Cannabis withdrawal is not life threatening 
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and resolves spontaneously without pharmacological intervention. Most symptoms appear within 
1 day of abstinence, peak within 2—3 days, and resolve within 1—2 weeks.
 
Resumption of cannabis use in CUD subjects who achieve abstinence: We expect that some of 
the CUD subjects will relapse to using cannabis after completing the study. To maximize the 
likelihood of continued abstinence, at the outset, subjects will be offered a referral to a cannabis 
treatment program at Yale.
2.3.2 Known Potential Benefits
Potential Benefits of the Proposed Research to the Subjects and Others
All study subjects with cannabis use will be referred for treatment at local treatment centers 
regardless of whether they choose to participate in and/or complete our research study. All 
participants in this study may derive subjective benefit from volunteering to take part in a study 
for the advancement of scientific knowledge.
 
Importance of the Knowledge to Be Gained
The knowledge to be gained regarding how cannabis use affects the brain is of extreme 
importance to better understand the negative and positive effects of cannabis. The study will 
produce data specific to cannabis use disorder and may guide the development of new 
treatments.
2.3.3 Assessment of Potential Risks and Benefits
Adequacy of Protection against Risk
 
Recruitment and informed consent All research subjects will be recruited under guidelines of 
the Yale University Institutional Review Board (Human Investigation Committee). The nature of 
the procedures, the risks, and financial remuneration for participation in the study will be 
discussed with each individual prior to obtaining informed written consent by a trained research 
assistant, postdoctoral fellow and/or the principal investigator of the study. The principal 
investigator is always available to address any questions that subjects may have during the 
consent procedure. Subjects are also "tested" on their knowledge about the study prior to study 
participation.
 
Protection against risk
1. Effective screening to exclude subjects who would be placed at a greater risk. This 
includes a medical history, the physical examination, and the screening studies (blood, 
urine and ECG) performed before starting studies. All screening procedures will be 
conducted by trained staff under the supervision of the PI.  Screening procedures may 
take place under HIC# 2000032181, in which case they will not be repeated if the 
participant is also enrolled in that study and has signed a release of information.
2. The investigator or a person designated by the investigator will explain the benefits and 
risks of participation in the study to each subject. Subjects will then be asked to explain 
the study in their own words to ensure their understanding of the study procedures prior 
to obtaining informed consent.
3.  To further prevent the risk of an adverse event and to minimize the harm incurred by 
such, all scans will be done in the presence of medical supervision and trained nursing 
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staff at the Yale PET center. In the event of serious medical complications, the PET 
center has immediate access to or consultation with specialized medical units at the Yale-
New Haven Hospital.
4. All subject information will be kept confidential and only members of the investigative 
team with appropriate IRB/HIC and HIPAA training will have access to the study data. 
Data will be maintained and secured in locked file cabinets or password protected 
electronic media. A numbering code will be used to assign a unique identifier to each 
subject. This information is available to study investigators and, in as far as the agents 
represent experimental pharmaceuticals, to representatives of the FDA and Nuclear 
Regulatory Commission for authorized inspection.
5. The dose of radiation is approved by the Yale University Radiation Safety Committee 
(RSC) and Radioactive Investigational Drug Committee (RIDC). All scans will be done 
in the presence of medical supervision and trained nursing staff in an institution 
specifically designed to support imaging studies. In the event of serious medical 
complications, the PET center staff have immediate access to or consultation with 
specialized medical units at the Yale-New Haven Hospital. Preparation of 
radiopharmaceuticals and performance of PET scans will be by radiochemists, 
physicians, and technologists of the Department of Radiology and Biomedical Imaging, 
Yale University School of Medicine. These professionals are qualified by training and 
experience in the safe use and handling of radiopharmaceuticals. Subjects will be asked 
about their previous radiation exposure and those who have had research exposure within 
the past year will be excluded if their cumulative annual exposure (including the present 
study) exceeds FDA limits. The information on the previous radiation exposure of study 
subjects will be notified to the study doctor.
6. No PET studies will be performed on pregnant or potentially pregnant women, as 
confirmed by pregnancy testing during evaluation and on each scan day before initiation 
of any scan procedure If subjects are breastfeeding they will not be able to participate in 
this research study.
7. The risks of bruising, clotting, and infection will be minimized by having venipuncture 
performed by trained and experienced personnel using aseptic technique to avoid injury 
due to fainting, the antecubital vein catheter will be inserted when the subjects are 
recumbent. The blood draws during PET scanning sessions will be obtained from the 
already inserted catheter, to minimize discomfort.
8. All subjects will be screened for any metallic objects that they may be holding or have 
implanted in their bodies using a questionnaire and all potential subjects with 
contraindications for MR will be exclude This questionnaire will be repeated 
immediately before each measurement to ensure that no metallic materials are brought 
into close proximity of the magnet, where they might be pulled toward the magnet. For 
additional security, subjects will be taken through a ferromagnetic metal detector 
immediately before going to the scan room.
9. The PI will review safety data, after every test day, during weekly research team 
meetings, and will suspend or modify the study (with IRB approval) if indicated.  The 
IRB will be duly informed if there are any reasons to warrant “holding” the study. A 
review of the study will be submitted to the IRB annually.
 
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3 - Objectives and Endpoints
3.1 Objectives and Endpoints
Aim 1: To determine differences in fronto-limbic mGluR5 availability between people with 
cannabis use disorder (CUD; n = 30) and healthy controls (HC; n = 30).  Subjects will 
undergo [18F]FPEB PET neuroimaging to determine mGluR5 availability. Hypothesis 1: Fronto-
limbic mGluR5 availability will be higher in individuals with CUD compared with HC. 
Aim 2: Within CUD subjects, to examine the associations between mGluR5 availability 
and: (a) CUD severity; (b) impulsivity; (c) EEG; and (d) neurocognition. CUD severity and 
impulsivity will be determined by validated instruments. EEG outcomes (e.g., theta power, LTP) 
known to be sensitive to both cannabis and glutamate will be collected during tasks of verbal 
memory22,23 and neural plasticity (long term potentiation; LTP).24  Hypothesis 2: Higher mGluR5 
availability will be associated with: (a) greater CUD severity (by DSM-5); (b) higher 
impulsivity; (c) lower theta-band power and LTP, and (d) worse performance on verbal memory.
Aim 3: Within CUD subjects, to determine changes in mGluR5 availability, EEG, and 
neurocognition following cannabis abstinence. CUD subjects from Aim 1 will repeat PET 
scanning, EEG, and cognitive testing following a 28-day period of confirmed abstinence aided 
by contingency management. Hypothesis 3: Following cannabis abstinence: (a) mGluR5 will 
decrease; (b) theta power/LTP will be increased; (c) memory will improve.
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4 - Study Design
4.1 Overall Design
4.2 Scientific Rationale for Study Design
A. SIGNIFICANCE 
A.1. Overview: How does cannabis affect glutamate receptors in the human brain?  
Cannabis has a wide range of health and behavioral effects, many of which remain incompletely 
understood.10 Yet, both recreational and medicinal use of the drug are widespread and continuing 
to increase in the US.  Cannabis is addictive. Nearly 30% of users may have cannabis use 
disorder (CUD), and nearly 12 million young adults used cannabis in 2019.5 Recreational use is 
now legal in 15 states, and medical use is legal in 35 states. As more states continue to legalize 
use for recreational purposes, cannabis use will likely continue to increase.25 This could result in 
an increasing number of people experiencing negative effects, including addiction.25
Cannabis use impairs cognition, memory, and motor coordination.  With more prolonged or 
heavy use, it increases the risk of developing schizophrenia or other psychoses.26  On the other 
hand, it is hypothesized that cannabis may have therapeutic effects in numerous neurologic 
conditions such as chronic pain, neurodegenerative diseases, and some psychiatric conditions 
(i.e. PTSD, anxiety, and sleep disorders). It is critical to more fully understand the neural 
mechanisms of cannabis to minimize harmful consequences and maximize therapeutic benefit.
Tetrahydrocannabinol (THC) is the primary psychoactive component of cannabis.  It is well-
established that many of the psychoactive and addictive properties of cannabis are mediated by 
the neural circuit involving THC binding to the cannabinoid receptor 1 (CB1), a presynaptic 
receptor that is widely distributed throughout the central nervous system.  Less well-understood, 
however, is the impact of CB1-THC binding on postsynaptic receptors such as metabotropic 
glutamate receptor 5 (mGluR5), a key regulator of glutamate homeostasis and 
endocannabinoid synthesis.11  There is strong evidence from the preclinical literature that 
cannabinoid exposure alters glutamate homeostasis via mGluR5 and CB1, which may 
mediate many important neural effects. Indeed, there is a close relationship between the 
cannabinoid and glutamatergic systems.19 However, this relationship has not been adequately 
studied in humans, and the degree to which disturbances in the glutamate system influences the 
potential harms and therapeutic benefits from cannabis use is unclear.  It is critical to advance 
our understanding of how chronic cannabis exposure affects the brain, including the 
consequences of these changes on neural function.  Importantly, we need to better understand 
whether neural dysregulation normalizes when cannabis use stops. This innovative multimodal 
(PET/EEG/neurocognitive) study will expand our knowledge and thus advance our 
understanding of the neurobehavioral mechanisms underlying cannabis addiction, with a focus 
on the metabotropic glutamate receptor 5 (mGluR5), in human subjects.    
A.2. Metabotropic Glutamate Receptor 5 (mGluR5): Importance in addiction and related 
behavioral phenotypes: Glutamate is the brain's primary excitatory neurotransmitter, and is 
critical for optimal neuronal function.27 Glutamate transmission between neural circuits linking 
key limbic and cortical brain regions maintains a balance between the reward-seeking behaviors 
necessary for survival and higher executive functions.28mGluR5, a key regulator of glutamate 
homeostasis, is a G protein-coupled receptor located mostly on post-synaptic neurons diffusely 
throughout the brain, especially key areas involved in addiction such as hippocampus, amygdala, 
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striatum, prefrontal cortex, and cingulate.29-31  It regulates synaptic plasticity of glutamatergic 
neurons, which is fundamental to learning and neural transmission of critical information.  
Alterations in mGluR5 receptor availability have been implicated in addiction32 and its related 
endophenotypes such as executive function impairment,33 mood disorders,34 and impulsivity.35  
Preclinical studies of mGluR5 knockout mice and/or the use of mGluR5 antagonists have 
revealed several key findings pointing to the likely involvement of mGluR5 in addictive 
disorders including: (1) reduced drug self-administration and reduced drug-seeking behavior;3637 
(2) attenuation of drug-induced rewarding effects38 and conditioned place preference39; and (3) 
reduced withdrawal.40,41 The precise mechanisms whereby these processes occur remain unclear. 
These data, combined with observations of mGluR5 effects on drugs of abuse in general,42,43 
make mGluR5 a potentially important molecular target in individuals with cannabis use 
disorder (CUD).
A.3. mGluR5: Importance in synaptic plasticity: Synaptic plasticity is critical for neuronal 
adaptations to a wide range of environmental conditions.  Local changes in synaptic strength 
[i.e., long-term potentiation (LTP) or depression (LTD)] underlie much of the brain's ability to 
change its physical structure in response to changes in the environment, particularly during 
learning and memory.  mGluR5 regulates synaptic plasticity of glutamatergic neurons, which 
is fundamental to learning and neural transmission of critical information.44,45  Activation of 
mGluR5 induces LTD in hippocampal area CA1 and cerebellar Purkinje neurons.46,47  Other 
preclinical studies have shown that antagonism of mGluR5 impaired LTP, spatial memory, and 
suppressed theta (5-10 Hz) activity in the dentate gyrus.17,18  Interestingly, the cannabinoid 
system is also implicated in learning, memory, and synaptic plasticity, indicating a potential 
convergence zone wherein mGluR5 and CB1 may interact functionally.48 Given the overlap 
between cannabis use, synaptic plasticity, learning, and cognition, it is critical to study 
mGluR5 in the context of cannabis use. 
Figure 1: mGluR5-CB1 relationship.  mGluR5 is a key regulator of glutamate homeostasis 
via cannabinoid receptor 1 (CB1).  The consumption of exogenous CB1 ligands via cannabis 
use may profoundly alter this process.  The focus of this grant is on how chronic cannabis use 
and abstinence specifically affects mGluR5 availability in humans with cannabis use disorder.  
Figure Source: Cristino, et.al., 2019 (See ref 11).
A.4. mGluR5: Key regulator of normal glutamate homeostasis via endocannabinoid 
synthesis: Under normal physiological conditions, preclinical models suggest that mGluR5 is a 
key regulator of glutamate homeostasis via CB1 at both excitatory (glutamatergic) and 
inhibitory (GABAergic) synapses, as shown in Figure 1 and described here.11  The focus of this 
grant is on understanding how chronic cannabis use and abstinence affect mGluR5 availability in 
humans with CUD. MGluR5 regulates glutamate primarily by triggering endocannabinoid (ECB) 
production.  ECBs (e.g., 2-AG) are naturally occurring molecules that regulate neurotransmitter 
release and synaptic plasticity by binding to presynaptic CB1. Under normal physiological 
conditions, stimulation of presynaptic excitatory neurons leads to glutamate release into the 
synaptic cleft and peri-synaptically.49 Glutamate binding to post-synaptic mGluR5 triggers the 
"on-demand" synthesis of the endocannabinoid 2-AG.  2-AG then binds in a retrograde manner 
to CB1, leading to synaptic depression and inhibition of further glutamate release.  Thus, under 
normal conditions, glutamate homeostasis is maintained via a negative feedback loop 
mediated by mGluR5 and endocannabinoid binding to presynaptic CB1.  This process may 
be disrupted by the consumption of exogenous cannabinoids.50
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Thus, mGluR5 is a critical, widespread molecular target involved in neural glutamate 
homeostasis that interacts directly with the cannabinoid system and is significantly involved in 
neural plasticity, memory, addiction, executive function, cognition, and emotional processing.  It 
is therefore critically important to study the relationship between CUD and mGluR5, and to 
relate this to neural function and behavioral outcomes related to addiction.  
A.5. Cannabis Use Disorder (CUD): Disruption of glutamate homeostasis via mGluR5 and 
CB1 (Aim 1): CUD is characterized clinically as a syndrome involving impaired control over 
cannabis use, problems occurring as a result of cannabis use, and signs of physiologic 
dependence (i.e. tolerance or withdrawal upon abstinence).51 Mechanistically, CUD is mediated 
primarily by recurrent and chronic THC binding to CB1.52  Cannabis and THC have widespread 
effects on neural glutamate transmission.50  Specifically, preclinical models have demonstrated 
that THC binding to CB1 inhibits glutamate release and leads to long-term synaptic depression 
of glutamatergic neurotransmission.53,54 Animal studies have demonstrated that after chronic 
exposure to THC and other CB1 agonists, there is a reduction in the number and function of CB1 
receptors.55-60 Furthermore, CB1 downregulation and desensitization has a distinct regional and 
temporal course, and is related to the duration and magnitude of exposure, with greater 
downregulation in cortical compared with subcortical regions.55,61,62 With prolonged abstinence, 
there is normalization in the number and function of presynaptic CB1 over 2 weeks, with faster 
recovery in subcortical compared to cortical regions.59  However, it is unknown what happens 
to postsynaptic mGluR5 from chronic cannabis use and following cannabis abstinence.  We 
plan to fill this knowledge gap through the proposed innovative and rigorous study.  As we 
illustrate in our robust preliminary data below (Sections C.1a., C.2.a., and C.3.a.), our central 
hypothesis is that mGluR5 upregulation occurs as a result of chronic cannabis use and may 
reverse upon cannabis abstinence.
A.6. Imaging cannabis use in humans (Aim 1): Imaging studies in humans have demonstrated 
two key findings regarding chronic cannabis use: (1) lower neural glutamate release14,15 and (2) 
CB1 downregulation,63,64 which has been shown to normalize after 4 weeks of abstinence.  These 
findings suggest changes in both CB1 number and function from chronic cannabis use.  In the 
first in vivo study of CB1 in cannabis users, Hirvonen et al., using positron emission tomography 
(PET) imaging and [18F]FMPEP-d2, demonstrated that chronic, heavy cannabis users showed 
~20% lower CB1 availability relative to controls.63 Consistent with animal studies, this reduction 
occurred in cortical but not in subcortical brain regions. In addition to showing lower CB1 
availability among people with chronic cannabis use, emerging data suggest that with abstinence 
of cannabis use, there is some recovery of CB1 receptor availability. We found that whereas 
there was a statistically significant reduction (~-15%) in CB1 availability in cannabis users at 
baseline relative to controls, these deficits persisted but were no longer statistically significant 
after 2 and 28 days of abstinence.  This suggests that there is some recovery of CB1 availability 
over time following cannabis abstinence.12 Collectively, these findings were interpreted as 
evidence of CB1 downregulation with chronic cannabis use that reversed with abstinence.
            A more recent study found lower mGluR5 availability in youth (ages 18-20 years) who 
were at-risk for substance use disorders (i.e., based on high impulsivity and aggression), 
including cannabis.65  No main effects of cannabis use alone on mGluR5 were observed.  
However, the study was limited by small subgroups (i.e., n = 3 CUD subjects) and uncertain 
timing and pattern of cannabis use relative to PET scanning.  Furthermore, the cross-sectional 
nature of the study limited the ability to understand dynamic changes in mGluR5 over time based 
on cannabis use status.  Our proposed study design, with scans performed longitudinally during 
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cannabis-maintained and cannabis-abstinent states in the same subjects, will help more 
definitively determine the relationship between chronic cannabis use and mGluR5.  
A.7. Central Hypothesis: Chronic Cannabis Use Increases mGluR5 Availability (Aim 1): 
The observation that chronic cannabis use leads to both lower synaptic glutamate release and 
CB1 downregulation raises a critical question: How do cannabis-induced changes in CB1 and 
glutamate release alter glutamate homeostasis?  We propose it occurs via increased mGluR5 
availability.  Normally, CB1 on presynaptic glutamatergic terminals are activated by retrograde 
endocannabinoids (i.e., 2-AG) that reduce Ca2+ influx, thus playing a modulatory role by 
inhibiting glutamate release (Figure 2, Left). While chronic cannabis exposure decreases the 
number of CB1 receptors (downregulation), constant heavy use of cannabis leads to tonically 
activated CB1 via THC (despite the known downregulation) (Figure 2, Right). This would 
result in decreased and unmodulated firing in glutamatergic pyramidal cell networks, thus 
leading to disruptions in functionally relevant neural activity (i.e., theta oscillations).19  We 
therefore propose that chronic cannabis use leads to less pre-synaptic glutamate release, less 
glutamate binding to mGluR5, and hence compensatory upregulation of mGluR5 (i.e., higher 
mGluR5 availability). Thus, it is clear that chronic exposure to THC from cannabis likely leads 
to profound disruption of glutamate homeostasis.  PET is the best imaging modality for 
studying changes in neuroreceptors in vivo and is the focus of Aim 1 of this proposal.  Using 
[18F]FPEB PET neuroimaging, we will measure mGluR5 availability in vivo in  people with 
cannabis use disorder (CUD) and compare with healthy controls. 
A.8. Cannabis Use Disorder: Insights from Electroencephalography (EEG) (Aim 2): While 
PET is the best imaging modality for studying changes in neuroreceptors in-vivo, this method is 
unable to provide information regarding how receptor changes affect neural function. By 
contrast, EEG is one of the few available neuroimaging methodologies that can directly 
measure neural events (postsynaptic potentials) with high temporal precision in humans. Thus, 
combining PET and EEG can be a powerful tool with which to examine the relationship 
between receptor dynamics and neural function.  EEG effects from chronic cannabis use, along 
with validated cognitive assessments, provide highly relevant clinical correlates to mGluR5 
availability.
The fact that CUD has disruptive effects across broad domains of brain function suggests a 
common neurophysiological mechanism. One potential mechanism may involve the role of CB1 
in modulating glutamate function, and hence the inhibitory-excitatory (I-E) balance of neural 
circuits. Skosnik et al. (2006) hypothesized that the neurobehavioral effects of cannabis use may 
involve alterations in neural synchronization.66 The impetus for this idea stems from the fact 
that interactions between glutamatergic pyramidal cells and GABAergic interneurons are 
involved in generating and maintaining neural oscillations in the theta and gamma (31-80 Hz) 
range.19,67-73 This may be particularly germane to the effects of cannabis, as synchronized neural 
oscillations are thought to be involved in memory and learning (domains of function that are 
known to be disrupted by cannabinoid agonists).  The effect of altered I-E balance may be 
particularly relevant to theta oscillations, as theta has been shown to be mediated in part by both 
CB1 and mGluR5 (as discussed above). From a functional standpoint, it has been established that 
theta may represent a "neural code" of learning and memory, and is related to synaptic 
plasticity.16,74,75  The relationships between mGluR5 availability, CUD severity, neural 
oscillations, and other clinically relevant domains (i.e., impulsivity, cognitive function) in people 
with CUD are the focus of Aim 2.  Using EEG, we now have the capacity to measure neural 
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function underlying memory and synaptic plasticity in human subjects in-vivo through 
theta band (4-7 Hz) neural oscillations and tetanic sensory stimulation, respectively.
A.9. Cannabis Use Disorder: Abstinence and the need for treatments (Aim 3): There is a 
substantial amount of misinformation regarding the addictive potential of cannabis (particularly 
on social media), leading to a general underappreciation of the issue among the public.76  
Estimates suggest that nearly 30% of cannabis users may have cannabis use disorder, and 10% 
will develop addiction (severe use disorder).7  Aim 3 focuses on the neural effects of prolonged 
(i.e., 4 weeks) cannabis abstinence in people with CUD.  It is critical to study CUD in human 
subjects longitudinally to advance our mechanistic understanding of disease pathogenesis and 
identify novel treatment targets.  Studying mGluR5, cognitive function, and EEG at two distinct 
timepoints in the addiction process (cannabis use and abstinence) will allow us to determine the 
long-lasting neurologic changes that might perpetuate CUD and influence disease severity, as 
well as changes occurring as a result of prolonged cannabis abstinence.
Lastly, it is worth noting that there are no current FDA-approved treatments for cannabis use 
disorder, despite the availability of several cannabinoid receptor agonists.  Aside from the 
known changes in CB1 number and function, the mechanisms underlying cannabis use 
disorder are still not fully understood and need further study.  Alterations in glutamate 
homeostasis are a likely contributing disease factor, given glutamate's central nature in 
endocannabinoid signaling.  mGluR5 likely plays an important role in this process.  
Developing our understanding of effects on glutamate homeostasis resulting from chronic use of 
cannabis and subsequent abstinence from the drug will advance our ability to develop new 
treatment targets and therapeutics for this population.  Collectively, these issues underscore the 
urgent need to understand the effects of chronic cannabis exposure on the brain, and importantly, 
whether these effects reverse when cannabis use stops.
A.10. Summary: Significance of the current proposal: Research efforts have been unable to 
keep pace with rapidly increased public demand for cannabis products, progressive legalization, 
and widespread use.  The preclinical literature provides a biologic basis for studying the effects 
of chronic cannabis use on the neural glutamate system.  However, it is critical to advance our 
understanding of cannabis use effects in human subjects.  Our preliminary data, described in 
further detail below (Sections C.1.a., C.2.a., and C.3.a.), indicate for the first time that mGluR5 
availability is significantly higher in cannabis users as compared with healthy controls. 
Among subjects who use cannabis chronically, mGluR5 availability may normalize following a 
period of cannabis abstinence.  Neural oscillations link directly with glutamatergic 
transmission and are disrupted from chronic cannabis use.  Finally, mGluR5 availability is 
strongly correlated with psychiatric symptoms and may predict disease severity in this 
population, indicating that mGluR5 is a clinically relevant biomarker of mental health in 
people with cannabis use disorder.  It is therefore both timely and innovative to study the effects 
of cannabis use on mGluR5 in humans with CUD using multimodal imaging (PET/EEG).  This 
study has the potential to increase our understanding of the mechanisms by which cannabis use 
affects the brain and is addictive.  It may lead to novel treatment targets among people who 
suffer from CUD.
B. INNOVATION
This proposal is highly innovative.  We propose the first known to us multimodal imaging study 
(combining PET, EEG, and cognitive testing) to measure the temporal course of changes in both 
the number and function of glutamate receptors in individuals with CUD at baseline and 
following a 4-week period of confirmed abstinence. By combining neuroreceptor PET imaging 
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(which is informative about changes in receptor availability) and EEG (which directly measures 
neural events with high temporal precision in humans), we will be able to examine the 
relationship between mGluR5 availability and neural function. In addition, examining 
correlations between changes in mGluR5 availability, CUD severity and related clinical features 
(i.e., impulsivity, craving, reward processing, etc.), electrophysiological indices sensitive to 
cannabinoids, and cognitive test performance in humans is highly novel. The proposed study 
may therefore uncover previously unknown consequences of cannabis use, and the data accrued 
from it will inform a large cross-section of researchers, including those studying cannabinoid 
dynamics, neural networks, addiction, and medicinal cannabis.  Insights from this study may 
shift treatment paradigms and lead to more targeted treatments for individuals with CUD. 
4.3 Justification for Dose
N/A
4.4 End of Study Definition
A participant is considered to have completed the study if he or she has completed all phases of 
the study including the last visit or the last scheduled procedure shown in the Schedule of 
Activities (SoA), Section 1.3.
The end of the study is defined as completion of the last visit or procedure shown in the SoA in 
the trial globally.
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5 - Study Population
5.1 Inclusion Criteria
A total of 60 subjects will be recruited: 30 with cannabis use disorder (CUD) and 30 healthy 
controls (HC).  Inclusion criteria for all subjects: 1) Age 18-55 years; 2) Voluntary, written, 
informed consent; 3) Physically healthy by medical history, physical, neurological, ECG and 
laboratory exams; 5) No personal or first-degree relative history of psychiatric disorders (outside 
of cannabis use for CUD group); 6) Full scale and verbal IQs > 80 (Wechsler Adult Intelligence 
Scale, Fourth Edition; WAIS-IV). CUD group: 1) Cannabis use disorder as determined by DSM-
5 structured interviews; 2) Urine toxicology evidence of cannabinoid use. Healthy control group: 
1) lifetime cannabis exposure less than 20 times, 2) no cannabis use in the past 2 years by self-
report, 3) a negative urine drug screen.  Controls will be matched to the CUD group by age, sex, 
socioeconomic status, education, nicotine use, and other relevant variables. 
5.2 Exclusion Criteria
Subjects will be excluded for the following reasons: 1) Other substance use disorder within 1 
year, except for nicotine; 2) Another primary DSM-5 Axis I major psychiatric disorder (e.g., 
schizophrenia, bipolar disorder, major depression, etc.) per SCID-5; 3) Urine toxicology results 
positive for other drugs such as opiates / opiate metabolites (e.g., methadone, buprenorphine, 
etc.); 4) A history of significant medical (cardiac, infectious, metabolic) or neurological illness 
(e.g., cerebrovascular disease, traumatic brain injury); 5) A history of seizures/epilepsy; 6) 
Medical contraindications to MRI imaging (e.g., ferromagnetic implants/foreign bodies, 
claustrophobia, etc.); 7) Pregnancy or breastfeeding (women).  After completing the informed 
consent process, subjects will have physical and neurological examinations and an 
electrocardiogram (ECG). Laboratory tests to be performed at screening to exclude medical 
illnesses include complete blood count (CBC) and differential, chemistries, kidney function tests 
[creatinine, blood urea nitrogen (BUN), urinalysis], liver function tests, sex hormones and 
thyroid stimulating hormone (TSH). Subjects will be excluded for major medical or neurological 
illness or laboratories consistent with these illnesses or suggesting contraindication to PET or 
MR imaging. A urine drug screen and a pregnancy test (for women) will be performed before 
both PET and MRI scans, and subjects excluded for positive screens. Exclusion is subject to PI 
discretion.
5.3 Lifestyle Considerations
Subjects will be required to undergo abstinence from cannabis for up to 4 weeks.  Subjects will 
receive motivational enhancement and contingency management (see payment structure in 
Human Studies Section) to promote and maintain abstinence. CUD subjects will undergo a 
second PET scan and EEG after 4 weeks of supervised abstinence.  CUD subjects will be 
required to come to the clinic two days a week for 1) motivational enhancement, 2) escalating 
contingency management (see Human Subjects section), 3) verbal confirmation of abstinence, 4) 
urinary drug testing to detect cannabis exposure in the past 1-24 hours, 4) supervised collection 
of urine samples to assay THC-COOH principal metabolite of THC. To account for dilution, 
urinary THC-COOH:creatinine (T:C) ratio will be calculated. Abstinence will be confirmed if 
the weekly means of urinary T:C ratio decreases over time and does not increase by more than 
50% relative to the prior specimen.87-89
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5.4 Screen Failures
Screen failures are defined as participants who consent to participate in the clinical trial but are 
not subsequently randomly assigned to the study intervention or entered in the study. A minimal 
set of screen failure information is required to ensure transparent reporting of screen failure 
participants, to meet the Consolidated Standards of Reporting Trials (CONSORT) publishing 
requirements and to respond to queries from regulatory authorities. Minimal information 
includes demography, screen failure details, eligibility criteria, and any serious adverse event 
(SAE).
Individuals who do not meet the criteria for participation in this trial (screen failure) because of a 
modifiable factor may be rescreened. Rescreened participants should be assigned the same 
participant number as for the initial screening.
5.5 Strategies for Recruitment and Retention
RECRUITMENT AND RETENTION PLAN 
Recruitment
All research subjects will be recruited under guidelines of the Yale University Institutional 
Review Board (Human Investigation Committee). Subjects will be recruited through a variety of 
advertising methods (study flyers, posters, internet/web postings, social media, radio, newspaper, 
and public access TV) as well as through referrals from other research groups/clinicians at the 
Yale-New Haven Hospital, Connecticut Mental Health Center, the CT VA, and/or the APT 
Foundation. Potential subjects will be encouraged to contact our study recruitment line for 
inclusion in our study. Subjects are recruited through our recruitment phone line. A member of 
our research staff will describe the study to participants who call, answer any questions the 
potential subjects have, and then complete a phone screening questionnaire to determine the 
subject's eligibility for an in-person screening visit. If an individual appears to meet enrollment 
criteria and is interested in participating, a face-to- face interview is conducted by the study staff 
and study physician. A release of information is obtained for review of any available historical 
and clinical data. A written authorization form is also obtained from each subject, permitting the 
research team to use, create, or disclose the subject's PHI for research purposes. The nature of the 
project, procedures, relative risks and benefits, and alternatives to participation in the project are 
discussed with the individual. Following this discussion, the individual is given a copy of the 
consent form to review, and any questions are answered. The process of informed consent will 
be obtained in accordance with local IRB standards by study personnel who have participated in 
institutionally approved training in human subject protection. Upon obtaining voluntary, written, 
informed consent, medical and psychiatric screening procedures will be used to confirm study 
eligibility. Subjects are free to discontinue their participation in the research at any time by 
requesting this verbally or in writing.
Retention
Retention of subjects with cannabis use disorder (CUD) will be maximized by our well-
established contingency management strategies and close contact with our study subjects through 
phone, e-mail, and in-person.   Using a combination of contingency management with escalating 
monetary rewards, counseling, and the use of frequent creatinine-adjusted quantification of 
urinary THC-COOH, we have been successfully able to have subjects remain abstinent for up to 
28 days with a relatively low drop-out rate (10%).
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Subjects will also be compensated for their time as both an inducement to participate and 
complete study procedures (by amounts / methods deemed appropriate and approved by our local 
IRB). 
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6 - Study Intervention
6.1 Study Intervention(s) Administration
6.1.1 Study Intervention Description
Study Schedule: 
Day 0: CUD subjects and healthy controls (HC) undergo PET scanning, an optional EEG, and 
cognitive testing. 
Days 1-27: CUD subjects abstain from cannabis use, aided by our validated contingency 
management strategy. 
Day 28: CUD subjects who are abstinent from cannabis use undergo repeat PET scanning, an 
optional EEG, and cognitive testing. 
Study Procedures: Eligible subjects will undergo an MRI scan for coregistration of PET data 
and for individualized source analysis of EEG. CUD subjects will be instructed not to use 
cannabis after midnight of Day 0, and last use of cannabis will be recorded. On Day 0, CUD 
subjects and HC will be scanned via PET, administered EEGs, and undergo cognitive testing. 
The same CUD subjects scanned on Day 0 will then be aided with cannabis abstinence and 
monitored over the next 4 weeks. Participants will be paid after completion of each of the study 
procedures they complete.  Payments include $450 for each PET scan, $50 for each MRI scan, 
$50 for each set of cognitive tests performed on or around Day 0 and 28, $50 for each optional 
EEG, and up to $1,100 if abstinence is successfully completed as confirmed by urine samples 
and report. 
Magnetic Resonance Imaging Session
MRIs will take place at the Magnetic Resonance Research Center (MRRC) at The Anlyan
Center (TAC) for Medical Research & Education, 300 Cedar Street, in New Haven. A member 
of the research staff will accompany the study participant to the MR scans and stay for the 
duration of the session.  
A high resolution, 3-dimensional Magnetization Prepared Rapid Acquisition Gradient Echo 
(MPRAGE) T1-weighted sequence will be used to acquire sagittal images for anatomical 
determinations and co-registration (TR=1500ms, TE=2.83ms, FOV=256x256mm2, matrix=256x 
256mm2, slice thickness=1.0mm without gap, 160 slices, voxel size 1.0 x 1.0 x 1.0 mm3). The 
structural MRI data will serve to guide placement of ROIs for PET and for conducting 
GMM/partial volume correction (PVC) analyses.  
We will also acquire rs-fMRI and DTI at both imaging time points, and these will be examined 
on an exploratory basis.    
If a recent MRI scan is available due to participation in another Yale HIC approved 
protocol,
the MRI scan session may not need to be completed for healthy controls.
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Positron Emission Tomography (PET) Sessions
PET procedures will be conducted at the Yale University PET Center. Female subjects of child 
bearing potential will be given a urine pregnancy test prior to the initiation of any imaging 
procedures. If the test is positive, the scan session will be canceled. HCs will complete a single 
PET scan session. CUD subjects will complete up to 2 PET scan sessions, one at baseline and 
one following cannabis abstinence.
PET scans are acquired as subjects lie supine on the scanner bed. Vital signs (blood pressure, 
pulse, respiration) will be obtained before and after radiotracer administration. A venous catheter 
will be used for IV administration of the radiotracer and for venous blood sampling. PET scans 
will be acquired using bolus plus constant infusion administration of ≤5 mCi of [18F]FPEB. 
Subjects are infused outside the scanner for the first 0-90 minutes during the pre-equilibrium 
period. Then, they will be scanned for 30 minutes in list-mode during the time the radiotracer 
reaches steady levels in the brain (90-120 minutes).
In the event that scans are not able to be completed as scheduled, subjects may be asked to return 
on another day to complete the scan. If a scan is canceled following radiotracer injection, 
subjects may receive an additional injection during their return visit, for a maximum of up to 2 
injections (HC) or 3 injections (CUD).
Cannabis Abstinence
On a weekly basis between Days 1-27, CUD subjects will be administered (1) the Withdrawal 
Discomfort score of the Cannabis Withdrawal Checklist, (2) the Cannabis Withdrawal Scale, 
which has subjective and objective (vital signs, weight) items, (3) Visual Analog Scale for Mood 
States, and (4) Pittsburgh Sleep Quality Index. CUD subjects will receive motivational 
enhancement and contingency management (CM) to promote and maintain abstinence. The 
escalating CM weekly payment schedule for cannabis abstinence is as follows: $50, $75, $125, 
$200, $650 (four-week abstinence bonus). If scheduling constraints extend the necessary 
abstinence period, an additional $200 per week of abstinence beyond 28 days will be paid. CUD 
subjects will be required to come to the clinic two days a week for (1) motivational 
enhancement, (2) escalating contingency management, (3) verbal confirmation of abstinence, (4) 
urinary drug testing to detect cannabis exposure in the past 1-24 hours, (5) supervised collection 
of urine samples to assay THC-COOH, the principal metabolite of THC. To account for dilution, 
urinary THC-COOH:creatinine (T:C) ratio will be calculated. Abstinence will be confirmed if 
the weekly means of urinary T:C ratio decreases over time and does not increase by more than 
50% relative to the prior specimen.[113-115] On Day 28, CUD subjects will undergo a second 
PET scan, EEG, and cognitive testing. 
The CUD subjects who relapse between the first and second scans (expected to be 10% of CUD 
subjects) will be: 1) considered dropouts and 2) referred for treatment if so desired. We will 
replace dropouts with new CUD subjects to ensure maintenance of adequate statistical power for 
hypothesis testing. We have accounted for dropouts in the analytic plan, study timeline, and 
budget. 
Cannabis Assessments: CUD severity is assessed using DSM-5 criteria.[51] Cannabis 
withdrawal is assessed by the Cannabis Withdrawal Scale.[116] Cannabis craving is assessed 
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using a 7-point scale. The Marijuana Problems Scale[117] will assess psychosocial impact of 
cannabis use. Multiple other clinically relevant features of CUD are described in the section 
below (Cognitive Testing and Behavioral Symptom Assessment). Detailed information on 
cannabis use patterns will be obtained using a time-line follow back approach: levels of cannabis 
consumption (estimated number of joints or edibles consumed) will be determined via interview 
for lifetime, the past six months, three months, one month, and then for the week prior to the test 
session as has been described previously [12,66,112,118,119]. Participants will be instructed to 
consider each day of the week and indicate, for an average week, how much they consumed per 
drug-use occasion for each length of time assessed. Cognitive Testing and Behavioral Symptom 
Assessment: The BIS/BAS will be used to assess trait impulsiveness and reward 
responsiveness.[120,121] Urgency, Premeditation (lack of), Perseverance (lack of), Sensation 
Seeking, Positive Urgency, Impulsive Behavior Scale will assess five impulsive personality 
traits. The Eriksen flanker task will supplement the assessment of the ability to suppress 
responses that are inappropriate to a particular context.[122] In addition, the Probabilistic 
Reward Task (PRT) will be used to provide an objective endophenotypic measure of reward 
responsiveness. 
On each of the scan days, CUD and controls subjects will also be administered 
neuropsychological tests from the CogState Cognitive Battery[123,124], which we previously 
showed to be sensitive to detecting cognitive deficits in mood disorders[125]: executive control 
(Groton Maze Learning task, GMLT; Set-Shifting task, SST and Go No-Go, GNG)[126-134] 
and working memory (One Back task, ONB, and Two Back task, TWB)[135-137]. Further, the 
battery has been well-validated (studies using CogState batteries have been published in over 
100 peer-reviewed journals).  Most important for the current study, this battery is ideal for 
within-subject designs (i.e., it was specifically developed for repeated administration with 
minimal practice or learning effects). Finally, the CogState has been shown to have high test-
retest reliability, thorough coverage of cognitive domains, comparable alternative forms, strong 
internal consistency, is well established in the general population, and has proven tolerability and 
acceptability. The entire battery takes approximately 30 minutes. For mood assessment, the 
Hamilton Depression Rating Scale (HAM-D) and Montgomery-Asberg Depression Rating Scale 
(MADRS) will be administered by highly trained and reliable raters. These assessments will be 
administered at screening and at the time of PET scans.
Additional Visits
In some situations, participants may be asked to come in for one or more additional visits. 
These visits may consist of one or more of the following, including repeat blood draw, repeat 
urine toxicology, assessments including for cannabis use, repeat MD evaluation/physical, repeat 
ECG, and other procedures listed in the screening visit. Repeat MRI and PET scans will not be 
performed at these visits. Participants will be compensated an additional $25 for each additional 
visit.
6.1.2 Dosing and Administration
The PET drug [18F]FPEB will be administered intravenously at a dose of no more than 5 mCi per 
scan day with a maximum of 1 scan for HC subjects and 2 scans for CUD.
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6.2 Preparation/Handling/Storage/Accountability
6.2.1 Acquisition and accountability
[18F]FPEB is provided by the Yale PET Center on the scheduled scan day. The radiochemistry 
laboratory is responsible for its preparation and release for administration. The remaining un-
injected radioactivity is returned to the radiochemistry quality control lab where it is stored in a 
lead-shielded pig until the sample completely decays (~10 half-lives) before it is discarded. 
6.2.2 Formulation, Appearance, Packaging, and Labeling
[18F]FPEB is formulated in saline for injection containing 0.9% ethanol and packaged in a sterile, 
clear, aluminum sealed top single dose glass (USP type I) vial with synthetic rubber stopper. The 
dose vial is labeled as described in the FDA approved Yale [18F]FPEB Drug Master File filed 
under IND application No. 155250. The final PET drug product solution is clear and colorless, 
with no visual evidence of cloudiness or particulate matter. 
6.2.3 Product Storage and Stability
[18F]FPEB is stored at room temperature in its respective sterile and nonpyrogenic dose vial and 
is stable for at least 8 hours after its preparation. 
6.2.4 Preparation
Under the supervisions of Dr. Henry Huang and Dr. Nabeel Nabulsi, [18F]FPEB is prepared by 
GMP trained radiochemistry production lab staff at the Yale PET Center immediately before 
administration. 
6.3 Measures to Minimize Bias: Randomization and Blinding
N/A
6.4 Study Intervention Compliance
CUD subjects will receive motivational enhancement and contingency management (CM) to 
promote and maintain abstinence.  The escalating CM weekly payment schedule for cannabis 
abstinence is as follows: $50, $75, $125, $200, $650 (four-week abstinence bonus).  We will 
collect urine samples to assay THC-COOH principal metabolite of THC. To account for dilution, 
urinary THC-COOH:creatinine (T:C) ratio will be calculated. Abstinence will be confirmed if 
the weekly means of urinary T:C ratio decreases over time and does not increase by more than 
50% relative to the prior specimen.113-115
The CUD subjects who drop out (estimated 10%) will be replaced with new CUD subjects to 
ensure maintenance of adequate statistical power for hypothesis testing.  We have accounted for 
dropouts in the analytic plan, study timeline, and budget. 
6.5 Concomitant Therapy
N/A
6.5.1 Rescue Medicine
N/A
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7 - Study Intervention Discontinuation and 
Participant Discontinuation/Withdrawal
7.1 Discontinuation of Study Intervention
Study participants may withdraw from the study at any time.  
7.2 Participant Discontinuation/Withdrawal from the Study
N/A
7.3 Lost to Follow-Up
A participant will be considered lost to follow-up if he or she fails to return for their scheduled 
visits and is unable to be contacted by the study site staff.
 The following actions must be taken if a participant fails to return to the clinic for a required 
study visit:

The site will attempt to contact the participant and reschedule the missed visit within 2 
weeks and counsel the participant on the importance of maintaining the assigned visit 
schedule and ascertain if the participant wishes to and/or should continue in the study.

Before a participant is deemed lost to follow-up, the investigator or designee will make 
every effort to regain contact with the participant (where possible, 3 telephone calls and, 
if necessary, a certified letter to the participant's last known mailing address or local 
equivalent methods). These contact attempts should be documented in the participant's 
medical record or study file.

Should the participant continue to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up.
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8 - Study Assessments and Procedures
8.1 Efficacy Assessments
OUTCOME
S
SOURCE/
MEASURE
HC
CUD 
(baseline)
Day 0
CUD (abstinence)
Day 28
Neural Receptors
 
mGluR5 
Availability 
PET ROI's 
VT: OFC, 
anterior 
cingulate, 
vmPFC, 
dlPFC, 
hippocampus
, amygdala
X
X
X
Cannabis Use Disorder — Clinically Relevant 
Functional Measurements
 
CUD 
Severity
DSM-5
 
X
 
Cannabis 
Use Patterns
Timeline 
Follow Back
 
X
X
Cannabis 
Withdrawal 
Severity
Cannabis 
Withdrawal 
Scale
 
X
X
Cannabis 
Craving
7-point Scale  
X
X
Cannabis-
Related 
Problems
Marijuana 
Problems 
Scale
 
X
 
Impulsivity
BIS/BAS
X
X
 
Reward 
Processing
Probabilistic 
Reward Task
X
X
X
Mood
HAM-D, 
MADRS, 
Visual 
Analog Scale 
X
X
X
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for Mood 
States
Sleep
Pittsburgh 
Sleep 
Quality 
Index
X
X
X
Cannabis Biomarkers
 
Cannabis 
Use Status
Urinary 
THC-
COOH*
X
X
X
Neural 
Function
 
 
 
 
Verbal 
Memory
EEG: Theta 
band 
power/coher
ence
X
X
X
Synaptic 
Plasticity / 
LTP
EEG: N100-
P200 
amplitude 
increase after 
auditory 
tetanus
X
X
X
Neurocogniti
ve Function
CogState 
Cognitive 
Battery
X
X
X
8.2 Safety and Other Assessments
Protection against risk
1. Effective screening to exclude subjects who would be placed at a greater risk. This 
includes a medical history, the physical examination, and the screening studies (blood, 
urine and ECG) performed before starting studies. All screening procedures will be 
conducted by trained staff under the supervision of the PI.  Screening procedures may 
take place under HIC# 2000032181.
2. The investigator or a person designated by the investigator will explain the benefits and 
risks of participation in the study to each subject. Subjects will be asked to complete a 
questionnaire to determine their understanding of the study procedures and risks, prior to 
obtaining written informed consent. The questionnaire consists of questions about the key 
risks (radiation, drug withdrawal, etc) and benefits of the study.
3. To further prevent the risk of an adverse event and to minimize the harm incurred by 
such, all scans will be done in the presence of medical supervision and trained nursing 
staff at the Yale PET center. In the event of serious medical complications, the PET 
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center has immediate access to or consultation with specialized medical units at the Yale-
New Haven Hospital.
4. All subject information will be kept confidential and only members of the investigative 
team with appropriate IRB/HIC and HIPAA training will have access to the study data. 
Data will be maintained and secured in locked file cabinets or password protected 
electronic media. A numbering code will be used to assign a unique identifier to each 
subject. This information is available to study investigators and, in as far as the agents 
represent experimental pharmaceuticals, to representatives of the FDA and Nuclear 
Regulatory Commission for authorized inspection.
5. The dose of radiation is approved by the Yale University Radiation Safety Committee 
(YU RSC). All scans will be done in the presence of medical supervision and trained 
nursing staff in an institution specifically designed to support imaging studi In the event 
of serious medical complications, the PET center staff have immediate access to or 
consultation with specialized medical units at the Yale-New Haven Hospital. Preparation 
of radiopharmaceuticals and performance of PET scans will be by radiochemists, 
physicians, and technologists of the Department of Diagnostic Radiology, Yale 
University School of Medicine. These professionals are qualified by training and 
experience in the safe use and handling of radiopharmaceuticals. Subjects will be asked 
about their previous radiation exposure and those who have had research exposure within 
the past year will be excluded if their cumulative annual exposure (including the present 
study) exceeds FDA limits. The information on the previous radiation exposure of study 
subjects will be notified to the study doctor.
6. No PET studies will be performed on pregnant or potentially pregnant women, as 
confirmed by pregnancy testing during evaluation and on each scan day before initiation 
of any scan procedur If subjects are breastfeeding, they will not be able to participate in 
this research study.
7. The risks of bruising, clotting, and infection will be minimized by having venipuncture 
performed by trained and experienced personnel using aseptic techni To avoid injury due 
to fainting, the antecubital vein catheter will be inserted when the subjects are recumbent. 
The blood draws during PET scanning sessions will be obtained from the already inserted 
catheter, to minimize discomfort.
8. All subjects will be screened for any metallic objects that they may be holding or have 
implanted in their bodies using a questionnaire and all potential subjects with 
contraindications for MR will be exclude This questionnaire will be repeated 
immediately before each measurement to ensure that no metallic materials are brought 
into close proximity of the magnet, where they might be pulled toward the magnet. For 
additional security, subjects will be taken through a ferromagnetic metal detector 
immediately before going to the scan room.
8.3 Adverse Events and Serious Adverse Events
8.3.1 Definition of Adverse Events (AE)
Adverse event means any untoward medical occurrence associated with the use of an 
intervention in humans, whether or not considered intervention-related (21 CFR 312.32 (a)).
8.3.2 Definition of Serious Adverse Events (SAE)
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An adverse event (AE) or suspected adverse reaction is considered "serious" if, in the view of 
either the investigator or sponsor, it results in any of the following outcomes: death, a life-
threatening adverse event, inpatient hospitalization or prolongation of existing hospitalization, a 
persistent or significant incapacity or substantial disruption of the ability to conduct normal life 
functions, or a congenital anomaly/birth defect. Important medical events that may not result in 
death, be life-threatening, or require hospitalization may be considered serious when, based upon 
appropriate medical judgment, they may jeopardize the participant and may require medical or 
surgical intervention to prevent one of the outcomes listed in this definition. Examples of such 
medical events include allergic bronchospasm requiring intensive treatment in an emergency 
room or at home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, 
or the development of drug dependency or drug abuse.
 
8.3.3 Classification of an Adverse Event
8.3.3.1 Severity of Event
For adverse events (AEs) not included in the protocol defined grading system, the following 
guidelines will be used to describe severity. 

Mild — Events require minimal or no treatment and do not interfere with the participant's 
daily activities. 

Moderate — Events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning.

Severe — Events interrupt a participant's usual daily activity and may require systemic drug 
therapy or other treatment. Severe events are usually potentially life-threatening or 
incapacitating.  Of note, the term "severe" does not necessarily equate to "serious".
8.3.3.2 Relationship to Study Intervention
All adverse events (AEs) must have their relationship to study intervention assessed by the 
clinician who examines and evaluates the participant based on temporal relationship and his/her 
clinical judgment. The degree of certainty about causality will be graded using the categories 
below. In a clinical trial, the study product must always be suspect.

Related — The AE is known to occur with the study intervention, there is a reasonable 
possibility that the study intervention caused the AE, or there is a temporal relationship 
between the study intervention and event. Reasonable possibility means that there is 
evidence to suggest a causal relationship between the study intervention and the AE.

Not Related — There is not a reasonable possibility that the administration of the study 
intervention caused the event, there is no temporal relationship between the study 
intervention and event onset, or an alternate etiology has been established.
OR

Definitely Related — There is clear evidence to suggest a causal relationship, and other 
possible contributing factors can be ruled out. The clinical event, including an abnormal 
laboratory test result, occurs in a plausible time relationship to study intervention 
administration and cannot be explained by concurrent disease or other drugs or 
chemicals. The response to withdrawal of the study intervention (dechallenge) should be 
clinically plausible. The event must be pharmacologically or phenomenologically 
definitive, with use of a satisfactory rechallenge procedure if necessary.
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
Probably Related — There is evidence to suggest a causal relationship, and the 
influence of other factors is unlikely. The clinical event, including an abnormal 
laboratory test result, occurs within a reasonable time after administration of the study 
intervention, is unlikely to be attributed to concurrent disease or other drugs or chemicals, 
and follows a clinically reasonable response on withdrawal (dechallenge). Rechallenge 
information is not required to fulfill this definition.

Potentially Related — There is some evidence to suggest a causal relationship (e.g., the 
event occurred within a reasonable time after administration of the trial medication). 
However, other factors may have contributed to the event (e.g., the participant's clinical 
condition, other concomitant events). Although an AE may rate only as "possibly related" 
soon after discovery, it can be flagged as requiring more information and later be 
upgraded to "probably related" or "definitely related", as appropriate.

Unlikely to be related — A clinical event, including an abnormal laboratory test result, 
whose temporal relationship to study intervention administration makes a causal 
relationship improbable (e.g., the event did not occur within a reasonable time after 
administration of the study intervention) and in which other drugs or chemicals or 
underlying disease provides plausible explanations (e.g., the participant's clinical 
condition, other concomitant treatments).

Not Related — The AE is completely independent of study intervention administration, 
and/or evidence exists that the event is definitely related to another etiology. There must 
be an alternative, definitive etiology documented by the clinician.
8.3.3.3 Expectedness
The Study PI will be responsible for determining whether an adverse event (AE) is expected or 
unexpected.  An AE will be considered unexpected if the nature, severity, or frequency of the 
event is not consistent with the risk information previously described for the study intervention.
8.3.4 Time Period and Frequency for Event Assessment and Follow-Up
The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to the 
attention of study personnel during study visits and interviews of a study participant presenting 
for medical care, or upon review by a study monitor.
All AEs including local and systemic reactions not meeting the criteria for SAEs will be captured 
on the appropriate case report form (CRF). Information to be collected includes event 
description, time of onset, clinician's assessment of severity, relationship to study product 
(assessed only by those with the training and authority to make a diagnosis), and time of 
resolution/stabilization of the event. All AEs occurring while on study must be documented 
appropriately regardless of relationship. All AEs will be followed to adequate resolution.
Any medical condition that is present at the time that the participant is screened will be 
considered as baseline and not reported as an AE. However, if the study participant's condition 
deteriorates at any time during the study, it will be recorded as an AE.
Changes in the severity of an AE will be documented to allow an assessment of the duration of 
the event at each level of severity to be performed. AEs characterized as intermittent require 
documentation of onset and duration of each episode.
The Study RA or PI will record all reportable events with start dates occurring any time after 
informed consent is obtained until 7 (for non-serious AEs) or 30 days (for SAEs) after the last 
day of study participation.  At each study visit, the investigator will inquire about the occurrence 
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of AE/SAEs since the last visit.  Events will be followed for outcome information until 
resolution or stabilization.
8.3.5 Adverse Event Reporting
Plans for Reporting Serious Anticipated and Unanticipated Adverse Events
Adverse events > level 3 will be reported to the IRB within 24 hours.  Other adverse events will 
be reported to the IRB in a timely manner, using the following predefined causal relationships:
 
Attribution of Risk Categories:
Definite:          Adverse event(s) will clearly be related to investigational agent(s) or other 
intervention
Probable:        Adverse event(s) will likely be related to investigational agent(s)
Possible:         Adverse event(s) may be related to investigational agent(s)
Unlikely:          Adverse event(s) will doubtfully be related to investigational agent(s)
Unrelated:       Adverse event(s) will clearly not be related to the investigational agents(s)
 
Monitoring for data integrity and safety will be the responsibility of the Principal Investigator, 
the Yale University IRB, and a Data and Safety Monitoring Board (DSMB). The DSMB will 
meet twice per year to review study progress. A DSMB will be used to provide additional 
protections for participants and to monitor progress on the study.
8.3.6 Serious Adverse Event Reporting
The study clinician will immediately report to the sponsor any serious adverse event, whether or not 
considered study intervention related, including those listed in the protocol or investigator brochure 
and must include an assessment of whether there is a reasonable possibility that the study 
intervention caused the event. Study endpoints that are serious adverse events (e.g., all-cause 
mortality) must be reported in accordance with the protocol unless there is evidence suggesting a 
causal relationship between the study intervention and the event (e.g., death from anaphylaxis). In 
that case, the investigator must immediately report the event to the sponsor.
All serious adverse events (SAEs) will be followed until satisfactory resolution or until the site 
investigator deems the event to be chronic or the participant is stable. Other supporting 
documentation of the event may be requested by the Data Coordinating Center (DCC)/study 
sponsor and should be provided as soon as possible.
The study sponsor will be responsible for notifying the Food and Drug Administration (FDA) of any 
unexpected fatal or life-threatening suspected adverse reaction as soon as possible, but in no case 
later than 7 calendar days after the sponsor's initial receipt of the information.  In addition, the 
sponsor must notify FDA and all participating investigators in an Investigational New Drug (IND) 
safety report of potential serious risks, from clinical trials or any other source, as soon as possible, 
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but in no case later than 15 calendar days after the sponsor determines that the information qualifies 
for reporting.
8.3.7 Reporting Events to Participants
Adverse events will be reported to participants individually.
8.3.8 Events of Special Interest
N/A
8.3.9 Reporting of Pregnancy (if applicable)
Positive pregnancy results will be reported to the study participant.
8.4 Unanticipated Problems
8.4.1 Definition of Unanticipated Problems (UP)
The Office for Human Research Protections (OHRP) considers unanticipated problems involving 
risks to participants or others to include, in general, any incident, experience, or outcome that 
meets all of the following criteria:

Unexpected in terms of nature, severity, or frequency given (a) the research procedures 
that are described in the protocol-related documents, such as the Institutional Review 
Board (IRB)-approved research protocol and informed consent document; and (b) the 
characteristics of the participant population being studied;

Related or possibly related to participation in the research ("possibly related" means there 
is a reasonable possibility that the incident, experience, or outcome may have been 
caused by the procedures involved in the research); and

Suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized.
Additional example text, applicable for device protocols:
This definition could include an unanticipated adverse device effect, any serious adverse effect 
on health or safety or any life-threatening problem or death caused by, or associated with, a 
device, if that effect, problem, or death was not previously identified in nature, severity, or 
degree of incidence in the investigational plan or application (including a supplementary plan or 
application), or any other unanticipated serious problem associated with a device that relates to 
the rights, safety, or welfare of subjects (21 CFR 812.3(s)).
8.4.2 Unanticipated Problem Reporting
The investigator will report unanticipated problems (UPs) to the reviewing Institutional Review 
Board (IRB) and to the Data Coordinating Center (DCC)/lead principal investigator (PI). The UP 
report will include the following information:
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
Protocol identifying information: protocol title and number, PI's name, and the IRB 
project number;

A detailed description of the event, incident, experience, or outcome; 

An explanation of the basis for determining that the event, incident, experience, or 
outcome represents an UP; 

A description of any changes to the protocol or other corrective actions that have been 
taken or are proposed in response to the UP.
To satisfy the requirement for prompt reporting, UPs will be reported using the following 
timeline:  
UPs will be promptly reported to the IRB of record and to the DCC/study sponsor in accordance 
with policy and regulatory requirements.  When Children's National serves as the IRB of record, 
UPs should be reported to the IRB within 7 business days of the investigator becoming aware of 
the UP.
8.4.3 Reporting Unanticipated Problems to Participants
Unanticipated problems will be reported to participants individually.
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9 - Statistical Considerations
9.1 Statistical Hypotheses
Aim 1: To determine differences in fronto-limbic mGluR5 availability between people with 
cannabis use disorder (CUD; n = 30) and healthy controls (HC; n = 30).  Subjects will 
undergo [18F]FPEB PET neuroimaging to determine mGluR5 availability. Hypothesis 1: Fronto-
limbic mGluR5 availability will be higher in individuals with CUD compared with HC. 
Aim 2: Within CUD subjects, to examine the associations between mGluR5 availability 
and: (a) CUD severity; (b) impulsivity; (c) EEG; and (d) neurocognition. CUD severity and 
impulsivity will be determined by validated instruments. EEG outcomes (e.g., theta power, LTP) 
known to be sensitive to both cannabis and glutamate will be collected during tasks of verbal 
memory22,23 and neural plasticity (long term potentiation; LTP).24  Hypothesis 2: Higher mGluR5 
availability will be associated with: (a) greater CUD severity (by DSM-5); (b) higher 
impulsivity; (c) lower theta-band power and LTP, and (d) worse performance on verbal memory.
Aim 3: Within CUD subjects, to determine changes in mGluR5 availability, EEG, and 
neurocognition following cannabis abstinence. CUD subjects from Aim 1 will repeat PET 
scanning, EEG, and cognitive testing following a 28-day period of confirmed abstinence aided 
by contingency management. Hypothesis 3: Following cannabis abstinence: (a) mGluR5 will 
decrease; (b) theta power/LTP will be increased; (c) memory will improve.
9.2 Sample Size Considerations
STATISTICAL DESIGN AND POWER
 
Frequency distributions and descriptive statistics for all variables will be computed prior to 
conducting analyses. If Shapiro-Wilk normality tests indicate non-normality for any continuous 
variable, we will compute necessary data transformations (e.g., logarithmic base 10) prior to 
conducting analyses. Of note, to conserve power, we will compute an average VT value across 
the 6 study ROIs to be used in all PET analyses. This choice is supported by the fact that 
preliminary data revealed large magnitude (i.e., r's>0.80-0.95) correlations between OFC, dlPFC, 
amygdala and hippocampus [18F]FPEB VT. Thus, we plan to treat this set of regions as a circuit 
and compute a mean composite score of [18F]FPEB VT for use in analyses.
 
Aim 1: To determine differences in fronto-limbic mGluR5 availability between people with 
cannabis use disorder (CUD; n = 30) and healthy controls (HC; n = 30).  Hypothesis 1: 
Fronto-limbic mGluR5 availability will be higher in individuals with CUD compared with HC.  
Power Calculations for Hypothesis 1: We approached power calculations by considering both 
our preliminary data and relevant and reliable literature (i.e., conducted with rigorous scientific 
methodologies, published in competitive, peer reviewed journals, with sufficient sample sizes) to 
determine the magnitudes of effect sizes that we might reasonably expect to observe in this 
study. No studies outside of our preliminary ones presented above (Research Strategy, Section 
C.1.a) have examined this topic of study specifically. However, we identified studies which 
examined differences in receptor availability in vivo between individuals who did and did not 
report use of cannabis using  PET.64,138-140 For example, a recent examination of CB1 receptor 
availability in cannabis users vs. non-users showed a mean VT 15% lower (d=-1.11)64. Another 
study found 20% lower CB1 in chronic daily cannabis users relative to non-users. Based on the 
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information provided in each manuscript, we calculated effect sizes for group differences. 
Observed effect sizes for group comparisons of cannabis users vs. non-users ranged from d= -
0.68 to -1.77, with a median of d=-1.11. These effect sizes are consistent with those observed in 
our preliminary data, which showed a large effect size associated with differences in mGluR5 
availability in CB relative to healthy control individuals in the fronto-limbic circuit (d= 1.72). 
We will evaluate hypothesis 1 (higher mGluR5 availability in cannabis users) using univariate 
analysis of variance (ANOVA). Assuming α=0.05 (two-tailed test) and two groups (cannabis vs. 
control), our proposed sample size of 60 (30 per group) will provide 81.07% statistical power to 
detect group differences as small as d= 0.82, which is smaller than the median effect size 
observed in previous literature and that observed in our preliminary data. Data Analysis for 
Hypothesis 1: As stated, we will conduct a univariate ANOVA to compare mean fronto-limbic 
[18F]FPEB VT between the HC and cannabis groups. Groups will be carefully matched on 
demographic variables. However, any demographic variables that differ significantly between 
groups, including notably age and sex, will be considered for entry as additional fixed 
factors (categorical variables) or covariates (continuous variables). A significant group 
difference suggestive of higher mGluR5 availability in CUD subjects will be considered 
supportive of Hypothesis 1. We will also conduct exploratory analyses to examine between-
group differences in mGluR5 in other regions, including those involved in affective and 
cognitive dysregulation (e.g., thalamus, striatum, insula), at the same significance level.
 
Aim 2: Within CUD subjects, to examine the associations between mGluR5 availability 
and: (a) CUD severity; (b) impulsivity; (c) EEG; and (d) neurocognition.  Hypothesis 2: 
Higher mGluR5 availability will be associated with: (a) greater CUD severity (by DSM-5); (b) 
higher impulsivity; (c) lower theta-band power and LTP, and (d) worse performance on verbal 
memory.  Power Calculations for Hypothesis 2: Using the same standards applied for hypothesis 
1, we considered our preliminary data and conducted a literature review to determine what 
magnitude of correlation we could reasonably expect to see in this study between relevant 
variables of interest. We included studies with similar methodologies, which reported 
correlations between similar variables (i.e. similar EEG measures and cognitive/behavioral 
constructs, and an in vivo measurement of neuroreceptor or transporter availability in humans 
using PET)141-145. Examples include an unpublished study from our research group in which the 
correlation between mGluR5 availability and impulsivity in BPD and PTSD individuals ranged 
from 0.46. to 0.80 in frontal and limbic brain regions of interest (n= 56). Observed correlations 
ranged in magnitude from 0.21 to 0.80 (positive or negative) with a median r = 0.57. Magnitude 
of relevant correlations observed in our preliminary data are comparable (i.e., correlation 
between impulsivity and fronto-limbic mGluR5 availability, r = 0.896). We will evaluate aim 2 
using correlational analyses. Using a correlation estimate of 0.55 (based on both review of the 
literature and our pilot data) and a two-tailed α =0.05, a priori power analysis suggested we will 
require a minimum n = 24 to achieve more than 80% power for the planned analysis. As we plan 
to conduct correlational analyses within the CUD group (n = 30), we will be adequately powered 
to test this hypothesis. Data Analysis for Hypothesis 2: Correlational analysis (Spearman or 
Pearson, as appropriate based on observed data distributions) will be used to test for associations 
between fronto-limbic mGluR5 availability, CUD severity, impulsivity, θ- band power and LTP, 
and working memory performance. Multiple regression analyses will also be considered to 
control for potential confounding variables. α will be set at 0.05 for all planned correlational 
analyses. We will also assess for associations between mGluR5 availability and performance on 
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other domains as secondary/exploratory analyses, keeping significance at the same level. 
Significant positive correlations between fronto-limbic mGluR5, CUD severity, and impulsivity 
will be supportive of Hypothesis 2. Likewise, significant negative correlations between fronto-
limbic mGluR5, θ- band power and LTP, and working memory performance will be considered 
supportive of hypothesis 2. Of note, the relationship between other relevant and clinically 
meaningful variables pertaining to CUD (e.g. craving, withdrawal, duration/intensity of use), and 
mGluR5 availability will also be examined in exploratory analyses.  The influence of potentially 
relevant demographic variables including age and sex will be examined.
 
Aim 3: Within CUD subjects, to determine changes in mGluR5 availability, EEG, and 
neurocognition following cannabis abstinence.  Hypothesis 3: Following cannabis abstinence: 
(a) mGluR5 will decrease; (b) theta power/LTP will be increased; (c) memory will improve.  
Power Calculations for Hypothesis 3: Using the same standards applied for Aims 1 and 2, we 
considered our preliminary data and conducted a literature review to determine the magnitude of 
effect sizes we might reasonably expect to observe with respect to change in mGluR5, EEG, and 
cognition as a function of abstinence from cannabis64,140,146,147. Values necessary to calculate 
associated effect sizes were not provided in the reviewed literature. For example, Hirvonen and 
colleagues (2012) observed a significant increase in CB1 receptor availability in cannabis users 
after abstinence in both the PFC and amygdala.  While percent change (pre-post abstinence) 
could be roughly estimated from the figure provided, exact values are not available, preventing 
effect size estimation.  We do not have preliminary data examining participants pre- and post- 
cannabis abstinence. However, we believe that comparison of available PET data showing in 
vivo mGluR5 availability in current cannabis users compared with former users may be 
indicative of the magnitude of change we can expect to see following abstinence. Comparison of 
mGluR5 availability in current and former cannabis users in our preliminary sample (n= 5 per 
group) showed a large effect (d = -1.2). We will evaluate Hypothesis 3 using repeated measures 
ANOVA. Using a moderate effect size estimate of d = 0.58 at α = 0.05 (highly conservative 
relative to the noted relevant finding in our preliminary data), a priori power analysis suggested 
we will require n = 30 to achieve 80.13% power for the planned analysis. Data analysis for 
Hypothesis 3: To test Hypotheses 3, we will conduct 3 repeated measures ANOVAs to examine 
changes pre- and post-cannabis abstinence in n = 30 CUD subjects. Specifically, models will 
evaluate: (1) changes in mGluR5 availability; (2) changes in θ-band power and coherence; (3) 
changes in cognition pre-post abstinence from cannabis in the CUD group. Variables that differ 
significantly between groups, including age and sex, will be considered for entry into the 
models. A statistically significant result indicative of lower mGluR5 availability following 
abstinence (model 1) or higher θ- band power and coherence (model 2) and improved cognition 
(model 3) will be considered supportive of Hypothesis 3.
9.3 Populations for Analyses
N/A
9.4 Statistical Analyses
9.4.1 General Approach  
See 9.2 above.
9.4.2 Analysis of the Primary Efficacy Endpoint(s)
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See 9.2 above.
9.4.3 Analysis of the Secondary Endpoint(s)
See 9.2 above.
9.4.4 Safety Analyses
9.4.5 Baseline Descriptive Analyses (if applicable)
9.4.6 Planned Interim Analyses (if applicable)
9.4.7 Sub-Group Analyses
9.4.8 Tabulation of Individual Participant Data
9.4.9 Exploratory Analyses
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10 - Supporting Documentation and Operational 
Considerations
10.1 Regulatory, Ethical, and Study Oversight Considerations
10.1.1 Informed Consent Process  
An IRB approved consent form describing the study procedures and risks will be given to the 
participant and written documentation of informed consent will be required prior to starting the 
study.
10.1.1.1 Consent/Assent and Other Informational Documents Provided to Participants  
Consent forms describing in detail the study intervention, study procedures, and risks are given 
to the participant and written documentation of informed consent is required prior to starting 
intervention/administering study intervention.  
10.1.1.2 Consent Procedures and Documentation  
Consent forms will be Institutional Review Board (IRB)-approved and the participant/legally 
authorized representative (LAR) will be asked to read and review the document. The research 
assistant, principal investigator, or co-investigator will explain the research study to the participant 
and answer any questions that may arise. This conversation will take place in a private room.  
Assent will not be conducted.  A verbal explanation will be provided in terms suited to the 
participant's comprehension of the purposes, procedures, and potential risks of the study and of their 
rights as research participants.  Participants/families/LAR will have the opportunity to carefully 
review the written consent form and ask questions prior to signing. The participants/family/LAR 
should have the opportunity to discuss the study with their family or surrogates, or think about it prior 
to agreeing to participate. The participant will sign the informed consent document prior to any 
procedures being done specifically for the study. Participants/families/LAR must be informed that 
participation is voluntary and that they may withdraw from the study at any time, without prejudice. A 
copy of the informed consent document will be given to the participants/families/LAR for their 
records. 
10.1.2 Study Discontinuation and Closure  
This study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause.  Written notification, documenting the reason for study suspension or 
termination, will be provided by the suspending or terminating party to study participants, 
investigator, funding agency, the Investigational New Drug (IND) or Investigational Device 
Exemption (IDE) sponsor and regulatory authorities.  If the study is prematurely terminated or 
suspended, the Principal Investigator (PI) will promptly inform study participants, the 
Institutional Review Board (IRB), and sponsor and will provide the reason(s) for the termination 
or suspension.  Study participants will be contacted, as applicable, and be informed of changes to 
study visit schedule.
Circumstances that may warrant termination or suspension include, but are not limited to:

Determination of unexpected, significant, or unacceptable risk to participants

Demonstration of efficacy that would warrant stopping  

Insufficient compliance to protocol requirements

Data that are not sufficiently complete and/or evaluable

Determination that the primary endpoint has been met
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
Determination of futility
Study may resume once concerns about safety, protocol compliance, and data quality are 
addressed, and satisfy the sponsor, IRB and/or Food and Drug Administration (FDA).]
10.1.3 Data Confidentiality and Participant Policy  
Participant confidentiality and privacy is strictly held in trust by the participating investigators, 
their staff, and the sponsor(s) and their interventions. This confidentiality is extended to cover 
testing of biological samples and genetic tests in addition to the clinical information relating to 
participants. Therefore, the study protocol, documentation, data, and all other information 
generated will be held in strict confidence. No information concerning the study or the data will 
be released to any unauthorized third party without prior written approval of the sponsor.
All research activities will be conducted in as private a setting as possible.
The study monitor, other authorized representatives of the sponsor, representatives of the 
Institutional Review Board (IRB), regulatory agencies or pharmaceutical company supplying 
study product may inspect all documents and records required to be maintained by the 
investigator, including but not limited to, medical records (office, clinic, or hospital) and 
pharmacy records for the participants in this study. The clinical study site will permit access to 
such records.
The study participant's contact information will be securely stored at each clinical site for 
internal use during the study. At the end of the study, all records will continue to be kept in a 
secure location for as long a period as dictated by the reviewing IRB, Institutional policies, or 
sponsor requirements.
Study participant research data, which is for purposes of statistical analysis and scientific 
reporting, will be transmitted to and stored in a secure electronic database such as REDCap. This 
will not include the participant's contact or identifying information. Rather, individual 
participants and their research data will be identified by a unique study identification number. 
The study data entry and study management systems used by clinical sites and by research staff 
will be secured and password protected. At the end of the study, all study databases will be de-
identified and archived.
Certificate of Confidentiality (if applicable)
To further protect the privacy of study participants, a Certificate of Confidentiality will be issued 
by the National Institutes of Health (NIH).  This certificate protects identifiable research 
information from forced disclosure. It allows the investigator and others who have access to 
research records to refuse to disclose identifying information on research participation in any 
civil, criminal, administrative, legislative, or other proceeding, whether at the federal, state, or 
local level. By protecting researchers and institutions from being compelled to disclose 
information that would identify research participants, Certificates of Confidentiality help achieve 
the research objectives and promote participation in studies by helping assure confidentiality and 
privacy to participants.
10.1.4 Future Use of Stored Human Specimens and Data  
Data collected for this study will be analyzed and stored in a secure electronic database such as 
REDCap. After the study is completed, the de-identified, archived data will be transmitted to and 
stored in the electronic database for use by other researchers including those outside of the study. 
Permission to store data will be included in the informed consent. 
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During the conduct of the study, an individual participant can choose to withdraw consent to have 
biological specimens stored for future research. However, withdrawal of consent with regard to 
biosample storage may not be possible after the study is completed. 
When the study is completed, access to study data and/or samples will be provided through the 
database.
10.1.5 Safety Oversight  
Data and Safety Monitoring Board (DSMB)
We will establish a DSMB to provide the highest protection for study participants. We will 
ensure that DSMB members collectively have expertise in neuroimaging (specifically PET and 
MRI) and cannabis/cannabinoids, which are most pertinent to subject risk for the proposed study.  
 
We will prepare twice yearly DSMB reports which include information on enrollment, 
participant retention rates, adverse events, and preliminary analyses as appropriate. The DSMB 
oversees an initial protocol review and then ongoing reviews. These reports are supplemented by 
rapid notification of all studies' serious adverse events. The PI will meet with the DSMB to 
review the study. The DSMB meets initially in an open session, attended by the PI and co-Is. 
Then, a closed session is held in which the DSMB Chair conducts a review of all issues and puts 
these to vote. The purview of the DSMB includes, but is not limited to, assessments of data 
quality and timeliness, participant recruitment, subject retention, safety and efficacy data, and 
protocol compliance. The DSMB also considers advances occurring elsewhere and their impact 
on the potential risks and benefits of the study. Following the DSMB meeting, a report of the 
meeting, including recommendations will be prepared and submitted to the PI, to the IRB, and to 
the funding agency. 
10.1.6 Key Roles and Study Governance
10.1.7 Clinical Monitoring
10.1.8 Quality Assurance and Quality Control  
Quality control (QC) procedures will be implemented beginning with the data entry system and data 
QC checks that will be run on the database will be generated. Any missing data or data anomalies 
will be communicated to the site(s) for clarification/resolution.
Following written Standard Operating Procedures (SOPs), the monitors will verify that the clinical 
trial is conducted and data are generated and biological specimens are collected, documented 
(recorded), and reported in compliance with the protocol, International Conference on Harmonization 
Good Clinical Practice (ICH GCP), and applicable regulatory requirements (e.g., Good Laboratory 
Practices (GLP), Good Manufacturing Practices (GMP)). 
The investigational site will provide direct access to all trial related sites, source data/documents, 
and reports for the purpose of monitoring and auditing by the sponsor, and inspection by local and 
regulatory authorities.
10.1.9 Data Handling and Record Keeping
10.1.9.1 Data Collection and Management Responsibilities  
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Data collection is the responsibility of the clinical trial staff at the site under the supervision of 
the site investigator. The investigator is responsible for ensuring the accuracy, completeness, 
legibility, and timeliness of the data reported.
All source documents should be completed in a neat, legible manner to ensure accurate 
interpretation of data. 
Hardcopies of the study visit worksheets will be provided for use as source document worksheets 
for recording data for each participant enrolled in the study.  Data recorded in the electronic case 
report form (eCRF) derived from source documents should be consistent with the data recorded 
on the source documents.
Clinical data (including adverse events (AEs), concomitant medications, and expected adverse 
reactions data) and clinical laboratory data will be entered into an a secure electronic database 
such as REDCap, a 21 CFR Part 11-compliant data capture system. The data system includes 
password protection and internal quality checks, such as automatic range checks, to identify data 
that appear inconsistent, incomplete, or inaccurate. Clinical data will be entered directly from the 
source documents.
10.1.9.2 Study Records Retention  
HHS funded study documents should be retained for a minimum of 3 years after the termination of 
the study.  Study documents should be retained for a minimum of 2 years after the last approval of a 
marketing application in an International Conference on Harmonization (ICH) region and until there 
are no pending or contemplated marketing applications in an ICH region or until at least 2 years 
have elapsed since the formal discontinuation of clinical development of the study intervention. 
These documents should be retained for a longer period, however, if required by local regulations. 
No records will be destroyed without the written consent of the sponsor, if applicable. It is the 
responsibility of the sponsor to inform the investigator when these documents no longer need to be 
retained.
10.1.10 Protocol Deviations  
A protocol deviation is any noncompliance with the clinical trial protocol, International 
Conference on Harmonisation Good Clinical Practice (ICH GCP), or Manual of Procedures 
(MOP) requirements. The noncompliance may be either on the part of the participant, the 
investigator, or the study site staff. As a result of deviations, corrective actions are to be 
developed by the site and implemented promptly.
These practices are consistent with ICH GCP:

4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3

5.1 Quality Assurance and Quality Control, section 5.1.1

5.20 Noncompliance, sections 5.20.1, and 5.20.2.
It is the responsibility of the site investigator to use continuous vigilance to identify and report 
deviations within 14 working days of identification of the protocol deviation, or within 30 
working days of the scheduled protocol-required activity.  All deviations must be addressed in 
study source documents, reported to NIDA Program Official and NIH.  Protocol deviations must 
be sent to the reviewing Institutional Review Board (IRB) per their policies. The site investigator 
is responsible for knowing and adhering to the reviewing IRB requirements. Further details about 
the handling of protocol deviations will be included in the MOP.
10.1.11 Publication and Data Sharing Policy  
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This study will be conducted in accordance with the following publication and data sharing policies 
and regulations:
National Institutes of Health (NIH) Public Access Policy, which ensures that the public has access to 
the published results of NIH funded research. It requires scientists to submit final peer-reviewed 
journal manuscripts that arise from NIH funds to the digital archive PubMed Central upon 
acceptance for publication.
This study will comply with the NIH Data Sharing Policy and Policy on the Dissemination of NIH-
Funded Clinical Trial Information and the Clinical Trials Registration and Results Information 
Submission rule. As such, this trial will be registered at ClinicalTrials.gov, and results information 
from this trial will be submitted to ClinicalTrials.gov. In addition, every attempt will be made to publish 
results in peer-reviewed journals.  Data from this study may be requested from other researchers 10 
years after the completion of the primary endpoint by contacting the study investigators.
10.1.12 Conflict of Interest Policy
The independence of this study from any actual or perceived influence, such as by the 
pharmaceutical industry, is critical.  Therefore, any actual conflict of interest of persons who have a 
role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed and 
managed. Furthermore, persons who have a perceived conflict of interest will be required to have 
such conflicts managed in a way that is appropriate to their participation in the design and conduct of 
this trial.  The study leadership in conjunction with the NIDA has established policies and procedures 
for all study group members to disclose all conflicts of interest and will establish a mechanism for the 
management of all reported dualities of interest.
10.2 Additional Considerations
10.3 Protocol Amendment History
10.4 Abbreviations
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LIST OF TABLES
Title
Section
Schedule of Activities (SoA)
2.2 Background
4.2 Scientific Rationale for Study Design
8.1 Efficacy Assessments
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