Neuroradiological and behavioral consequences of repeated ... · research. I’d like to sincerely thank my faculty mentor, Craig Ferris, for giving me the opportunity and guidance

Northeastern University

Undergraduate Honors Thesis

Neuroradiological and behavioral consequences of repeated inhaled

cannabis exposure: an exploratory, sex-based study in adolescent mice

First Author: Faculty Mentor:

James R. Coleman, Jr. Craig Ferris, PhD

Candidate for BS in Behavioral Neuroscience Department of Psychology

Northeastern University, Class of 2020 Northeastern University

[email protected] [email protected]

A thesis submitted in fulfilment the requirements

for the degree of Bachelor of Science

in the

Center for Translational Neuro-Imaging

Department of Psychology

Northeastern University

April 13, 2020

Co-Authors: Coleman, JR1, Madularu, D1, Athanassiou, M2, Knudsen, A1, Alkislar, I1, Cai, X1, Kulkarni, PP1, Ferris,

CF1

1Center for Translational Neuro-Imaging, Northeastern University, Boston, MA 2McGill University, Montreal, QC, Canada

Coleman et al., 2020

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PREFACE

The foundation for this research project was laid during my time spent on co-op in the

Center for Translational Neuro-Imaging after being awarded the Schafer Research Co-op

Scholarship in 2018. I started my co-op shortly after Dr. Ferris received approval from the

federal government to perform research on cannabis and its effects on the central nervous system

in rodent models during a particularly relevant time (MA legalized recreational marijuana in

2016, while dispensaries opened their doors to adults 21+ in 2018). I continued to work on the

project after my co-op ended through a directed study in Spring 2019 and completed my thesis

during my final semester in Spring 2020. I’ve been honored to present my work in posters at

Northeastern’s RISE 2019 and the Society for Neuroscience 2019 conference in Chicago.

Additionally, this work has been submitted for publication in the European Journal of

Neuroscience (EJN) and is currently under review.

In truth, this research project would not have been possible without the support of so

many people who consistently believed in me, the work I’ve done, and the value in this line of

research. I’d like to sincerely thank my faculty mentor, Craig Ferris, for giving me the

opportunity and guidance which led to my success as a undergraduate researcher, Dan Madularu,

for his mentorship and support in writing my final draft, and all of my other co-authors for their

work in data collection and analysis of this ambitious project. A huge thanks to Dr. Andrew

Schafer, who’s generous funding made my first co-op at the CTNI possible. I’d also like to thank

my parents, Denise and James, for their support in shaping who I am today, as well as my friends

for being a source of joy and laughter for me throughout this long process.


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Abstract

Social norms and legality surrounding the use of medical and recreational marijuana

are changing rapidly both in the US and abroad. With the recent wave of legalization efforts, it is

urgent that clinically translatable research is conducted so that we may better understand this

drug’s long-term effects on the central nervous system. The aim of this study was to assess any

sex-based in the effects of chronically inhaled vaporized cannabis on the adolescent male and

female mouse CNS using non-invasive multimodal magnetic resonance imaging (MRI) and the

novel object preference (NOP) behavioral task. Male and female mice chronically exposed to

inhaled cannabis show sex-specific differences in resting-state functional connectivity (rsfcMRI),

voxel based morphometry (VBM), and diffusion weighted imaging (DWI) as compared

to animals of the same sex exposed to placebo. Furthermore, male mice exposed to inhaled

cannabis failed to investigate a novel object beyond chance during a novel object preference test.

Results are discussed in the context of clinical translatability and sex difference in the inhaled

cannabis space.


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Introduction

As it is the case worldwide, the use of marijuana in Canada and the United States is

prevalent and it is expected to increase with its legalization. As evidence of the therapeutic

potential of marijuana increases, so too will its use for medical purposes. Marijuana is no longer

considered the innocuous “soft drug” of yesteryear, as the potency of the different marijuana types

has tripled over the past 30 years, with levels of tetrahydrocannabinol increasing from 3.5% to

12.6%; more potent marijuana strains (18-22% tetrahydrocannabinol) are not uncommon. The

average age of first-time marijuana users is 17 years, a crucial neurodevelopmental stage when

brain structures (i.e. prefrontal cortex) responsible for executive function (i.e. working memory,

planning) have yet to reach full maturity (Cuttler et al., 2007).

The marijuana plant has two primary chemical constituents with neurobiological activity,

Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), acting through the CB1 receptor that is

found throughout a wide number of brain areas including the dorsal and ventral striatum,

hippocampal complex and prefrontocortical areas receptors (Herkenham et al., 1991; Tsou et al.,

1998; Tsou et al., 1999; Pickel et al., 2004). Clinical neuroradiological and cognitive studies link

chronic cannabis exposure with increases in cerebellar gray matter, alteration of cerebellar resting

state activity and cognitive deficits (for review, see Batalla et al., 2013; Blithikioti et al., 2019).

Differences in hippocampal sizes were also reported in response to chronic cannabis use compared

to control, with heavy cannabis users showing significantly smaller left and right hippocampi

volume (Yucel et al., 2008; Ashtari et al., 2011). On the other hand, Weiland and colleagues failed

to find even a modest difference in hippocampus morphometry between chronic users and controls

(2015); similar results (or lack thereof) were reported by Tzilos and colleagues after studying a

group of long-term cannabis users (Tzilos et al., 2005). Finally, continued heavy cannabis use had


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no effect on hippocampal volumes in young adults (18-24 years old) compared to controls, as

reported recently by Koenders et al. (2017).

The source of the discrepancies between findings in the clinical literature may be linked to

a variety of factors and unknowns such as THC concentration, THC/CBD characteristics of the

used cannabis, pharmacological interactions between cannabinoids and tobacco, to name a few.

These shortcomings can be circumvented in preclinical studies (although at the cost of reduced

clinical translatability), with an increasing numbers of studies on adolescent rodents consistently

reporting long-term deficits in cognition following repeated exposure to injected or infused THC

(Fehr et al., 1976; Rubino et al., 2012; Zamberletti et al., 2012; Silva et al., 2016; Kasten et al.,

2017; Murphy et al., 2017). Chronic THC administration is reported to impair radial arm maze

performance in rodents (Fehr et al., 1976; Stiglick & Kalant, 1982), with evidence of lasting

neuronal atrophy and decreased synaptic density observed in in the CB1 receptor-rich hippocampal

CA3 region after 3 months of oral THC administration (Fehr et al., 1976). Chronic THC

administration has also been shown to cause longer-lasting memory deficits in adolescent rats

compared to adults (Scallet et al., 1987). More recent preclinical studies have adopted more

clinically-translatable methodologies, focused on the effects of inhaled cannabis and cannabis and

cannabis extracts, with Blaes and colleagues (2019) showing that although acute exposure to

cannabis smoke (or placebo) had no cognitive outcomes in adult male rats, it had an enhancing

effect on working memory on the female group; this group showed a lower baseline pre-treatment.

The same study showed that acute administration of either THC or the CB1 antagonist rimonabant

impaired working memory performance, pointing at a distinct difference in cognitive outcome vis-

à-vis route of administration; similar findings were reported by Manwell et al. (2014). Niyuhire

and colleagues (2007) found that both inhaled cannabis, as well as injected THC had a disruptive


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effect on platform location acquisition in mice and the recall of the platform location once it was

learned; these effects were blocked by rimonabant, a selective CB1 receptor blocker. Finally, Freels

and colleagues (2020) showed recently that vaporized cannabis extracts have reinforcing

properties and support conditioned drug-seeking behavior in rats.

Although a number of preclinical studies investigated the cognitive and physiological

effects of THC and inhaled cannabis, there is a lack of preclinical neuroradiological data

paralleling the clinical studies. To this end, the current study was designed to address the following

questions, in a clinically translatable manner: i) does prolonged exposure to inhaled cannabis alter

functional connectivity and volumetric measurements in mice, ii) are these differences

accompanied by behavioral deficits, and iii) are there any behavioral and/or neuroradiological sex

differences in response to inhaled cannabis in mice?

Materials and Methods

Subjects

Male and female C57BL/6 mice (N=32) were ordered from The Jackson Laboratory (Bar

Harbor, ME) and were received at age PND21. Animals were group housed in clear plastic cages

(4 per cage) with ad libitum food and water. Cages were stored in a temperature-controlled

vivarium on a 12-h light/dark cycle. Animals were divided into 4 experimental groups: male

+ cannabis (n=8), female + cannabis (n=8), male + placebo (n=8), and female + placebo (n=8). All

subjects were cannabis-naïve before exposures began on PND23 (adolescence). Experimental

procedures used followed NIH guidelines and were approved by the Institutional Animal Care and

Use Committee (IACUC) at Northeastern University.


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Drug

Cannabis sativa, containing 10.3% THC and 0.05% CBD, and placebo cannabis containing

0.0%THC and 0.0% CBD was acquired from the National Institute on Drug Abuse (NIH/NIDA,

Bethesda, MD) through the Research Triangle Institute (Research Triangle Park, NC).

Cannabis exposure.

Groups of animals were placed in a 38-L exposure chamber (60cm x 45cm x 20cm),

modified from a “41 qt. Weathertight Tote Clear” (The Container Store, Chestnut Hill, MA). In

order to ensure consistent expose, a plastic divider was used to split the area of the exposure

chamber into two halves, so that two groups could be exposed simultaneously in the same session:

male and female cannabis or male and female placebo (16 animals per session). Subjects were

acclimated to the exposure environment for two days prior to exposure to reduce any stress of the

novel environment. A Volcano Vaporizer (Storz and Bickel, Tuttlingen, Germany) was used to

heat cannabis plant material below the point of complete combustion in order to vaporize the active

ingredient (THC), minimizing the generation of harmful free radicals such as polycyclic aromatic

hydrocarbons (PAHs) associated with the combustion of organic plant material. The vaporizer was

preheated approximately 210°C and loaded with 0.450g of pulverized cannabis plant material. We

chose this weight at this concentration based on preliminary data (Supplementary Fig. 1) showing

that this approach yielded similar serum THC concentrations to those reported in human users (i.e.

130-150 ng/ml; Huestis et al., 2007).

Tubing was attached between the vaporizer and the exposure chamber and the heating fan

was run for 60-s, filling the exposure chamber with vaporized marijuana aerosols. After 30

minutes, mice were removed from the exposure chamber and returned to their cages. This exposure

protocol occurred daily for 28 consecutive days.


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Imaging system

Imaging took place 2 days after exposure cessation, while animals were age PND53 (adult).

Imaging experiments were performed on anesthetized mice (1-2% isoflourane). A detailed

description of the mouse imaging system is published elsewhere (Ferris 2014). Notably, we used

a quadrature transmit/receive volume coil (ID = 38 mm) that provided excellent anatomical

resolution and signal-noise-ratio for voxel-based blood oxygen level dependent (BOLD) fMRI.

Furthermore, the unique design of our mouse holder (Animal Imaging Research; Holden, MA)

essentially stabilized the head in a cushion, minimizing any discomfort caused by other ear bars

and pressure points that are commonly used to immobilize the head for awake animal imaging

(Ferris et al., 2014).

Image acquisition and pulse sequence

Experiments were conducted using a Bruker Biospec 7.0T/20-cm USR horizontal magnet

(Bruker; Billerica, MA) and a 20-G/cm magnetic field gradient insert (ID = 12 cm) capable of a

120-µsec rise time. At the beginning of each imaging session, a high-resolution anatomical data

set was collected using the RARE pulse sequence (18 slices; 0.75 mm; FOV 1.8 cm; data matrix

256 x 256; TR 2.1 sec; TE 12.4 msec; Effect TE 48 msec; NEX 6; 6.5 min acquisition time).

Functional images were acquired using a multi-slice Half Fourier Acquisition Single Shot Turbo

Spin Echo (HASTE) pulse sequence (18 slices; 0.75 mm; FOV 1.8 cm; data matrix 96 x 96; TR 6

sec; TE 4 msec; Effect ET 24 msec; 15 min acquisition time; in-plane resolution 187.5 µm2). Each

functional imaging session consisted of uninterrupted data acquisitions (whole brain scans) of 150

scan repetitions for a total elapsed time of 15 min.

Imaging data analysis

The effect of vaporized cannabis inhalation on brain activity was quantified through


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positive and negative percent changes in BOLD signal relative to baseline. The statistical

significance of these changes was assessed for each voxel (~15,000 per subject, in their original

reference system) via independent Student t-tests, with a 2% threshold to account for normal

fluctuations of BOLD signal in the anesthetized rodent brain (Brevard 2013). As a result of the

multiple t-tests performed, a false-positive detection controlling mechanism was introduced

(Genovese 2002), to ensure that, on average, the false positive detection rate remained below 0.05.

The following formula was used:

𝑃𝑖 ≤ 𝑖

𝑉

𝑞

𝑐(𝑉) ,

where 𝑃𝑖 is the p-value from the t-test at the 𝑖-th pixel within the region of interest (ROI) containing

𝑉 pixels, each ranked based on its probability value. The false-positive filter value 𝑞 was set to 0.2

for our analysis, and the predetermined constant 𝑐(𝑉) was set to unity (Benjamini and Hochberg

1995), providing conservative estimates for significance. Pixels that were statistically significant

retained their relative percentage change values, while all other pixel values were set to zero. A

95% confidence level, two-tailed distributions, and heteroscedastic variance assumptions were

employed for the t-tests.

Voxel-based percent changes in BOLD signal were combined across subjects within the

same group to build representative functional maps. To this end, all images were first aligned and

registered to a 3D Mouse Brain Atlas© with 139 segmented and annotated brain regions (Ekam

Solutions; Boston, MA). The coregistrational code SPM8 was used with the following parameters:

Quality: 0.97, Smoothing: 0.35 mm, Separation: 0.50 mm. Gaussian smoothing was performed

with a FWHM of 0.8 mm. Image registration involved translation, rotation, and scaling,

independently and in all three dimensions. All applied spatial transformations were compiled into


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a matrix [𝑇𝑗] for the 𝑗-th subject. Every transformed anatomical pixel location was tagged with

major and minor regions, to generate fully segmented representations of individual subjects within

the atlas.

Next, composite maps of the percent changes in BOLD signal were built for both groups.

Each composite pixel location (row, column, and slice) was mapped to a voxel of the 𝑗-th subject

by virtue of the inverse transformation matrix [𝑇𝑗]−1. A tri-linear interpolation of subject-specific

voxel values determined their contribution to the composite representation. The use of the inverse

matrices ensured that the full composite volume was populated with subject inputs. The average

of all contributions was assigned as the percent change in BOLD signal at each voxel within the

composite representation of the brain. The number of activated voxels in each of the 139 regions

was compared between the control and cannabis groups using a Mann-Whitney U test. Brain

regions were considered statistically different between experimental groups when p 0.05.

Novel Object Preference Assay

Novel object preference (NOP) was assessed in an arena constructed of white PVC,

measuring 35 cm × 35 cm × 35 cm. NOP was conducted 2 months after exposure cessation

(animals age PND100) in order to see if any differences in behavior would persist into adulthood.

A video camera was mounted above the arena to record familiarization and test phases for

subsequent analysis. The test stimuli were objects made of plastic figurines, and varied in color,

height and width (i.e. 3–5 cm), attached onto the floor using Velcro, at 10 cm from opposing

corners. There were three identical copies of each object, which were used interchangeably, and

washed after each use with water. Animals were tested on the novel object preference task


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The protocol and analysis of NOP data was adapted from Madularu and colleagues (2014).

Prior to this testing trials, each mouse was given the opportunity to habituate to testing conditions

during one daily 15-min session over three consecutive days, in which mice were allowed to

investigate two objects in the same arena where subsequent testing occurred; the two objects

encountered during the habituation session were not used for NOP testing. Each session was video-

recorded and object preference was quantified as time investigating the novel object divided by

total time investigating both objects for the first minute of the TEST session. The short sessions

also consider the fact that mice placed in a novel environment are most likely to investigate in the

first 5 min, followed by periods of relative inactivity.

Investigation ratios (IR = time spent investigating novel object / time spent investigating

both obects) during the first minute of the 5-min TEST session were assessed using single-sample,

two-tailed t-tests, and performance was compared to chance (i.e., IR = 0.5). Total time spent with

both objects during the 5-min FAM sessions also was recorded and analyzed using dependent

samples t-tests. An investigation ratio significantly greater than 0.5 indicates that the voles were

spending more time with the novel object. Conversely, a ratio significantly smaller than chance

was used as an index of a preference for the familiar object.

Results

Resting-state functional connectivity MRI (rs-fcMRI)

As shown in Fig. 1, significant differences in the resting state functional connectivity (rs-

fcMRI) signal between several distinct brain networks were observed in both male and female

groups exposed to inhaled cannabis when compared to control subjects of the same sex. Males are

shown to have strong hypoconnectivity, or negative connectivity, between the raphe nuclei and


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several limbic areas such as the thalamus (ventral, posterior, and parafasicular) and the

hippocampus as assessed by BOLD response. Hypoconnectivity between the hypothalamus/

thalamus and bed nucleus of stria terminalis (BNST) and the temporal cortex is also prevalent in

males exposed to inhaled vaporized cannabis. This pattern of hypoconnectivity is not observed in

control males nor in either the female groups. Both males and females in the cannabis exposure

group showed a decrease in functional connectivity compared to controls in several brain regions

such as the substantia nigra/VTA connections to the midbrain, CA1/CA3 connections to the

thalamus, as well as the raphe/pons connections to the parabrachial/trigeminal area.

Voxel-based Morphometry

Female mice exposed to inhaled cannabis showed significant differences in brain volume

in 17 ROIs compared to female controls, while the male + cannabis group showed significant

differences in 5 ROIs compared to control males (Fig. 2). Regions significantly reduced in volume

in females chronically exposed to inhaled cannabis (11/17 sig. ROIs) include the parietal cortex (p

= 0.002), locus coeruleus (p = 0.009), 4th cerebellar lobule (p = 0.024) and ventral tegmental area

(p = 0.035), while areas increasing in volume (6/17 sig. ROIs) include the olfactory tubercles (p =

0.005), lateral dorsal thalamus (p = 0.0037), paraventricular hypothalamus (p = 0.040), and anterior

cingulate cortex (p = 0.049). Males chronically exposed to inhaled cannabis showed substantially

fewer changes which include decreases in volume of the central amygdaloid area (p = 0.006),

ambiguus area (p = 0.031), caudate putamen (p = 0.036), medial amygdaloid area (p =0.040), and

superior colliculus (0.046).


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Diffusion Weighted Imaging (DWI) with Quantitative Anisotropy

Chronic exposure to inhaled marijuana during adolescence affects white matter

microarchitecture in both females and males as shown by differences in apparent diffusion

coefficient (ADC) between placebo and marijuana groups for each sex (Fig. 3). Males and females

shared significant changes in ACD in 23 areas including midbrain structures such as the superior

colliculus (F: p = 0.002 / M: p = 0.024,), nucleus accumbens core (F: p = 0.049 / M: p = 0.002),

dorsal raphe (F: p = 0.005 / M: p = 0.015), locus coeruleus (F: p = 0.009 / M: p = 0.024), and

periaqueductal gray (F: p = 0.006 / M: p = 0.004). Several areas associated with the limbic system

are also significantly affected in males and females, specifically the subiculum (F: p = 0.011 / M:

p = 0.027), dentate gyrus (F: p = 0.013 / M: p = 0.035), insular rostral cortex (F: p = 0.028 / M: p

= 0.006), and ventral pallidum (F: p = 0.015 / M: p = 0.001). The cerebellum, associated with

motor control and planning, had decreases in ACD compared to control, specifically in the

cerebellar nuclear area (F: p = 0.003 / M: p = 0.001) and several cerebellar lobules (3rd, 4th, 5th

and 7th). Females exposed to chronic inhaled cannabis show distinct changes in ACD in areas

including the dorsal hippocampal commissure (p = 0.001), orbital cortex (p = 0.009), medial

geniculate (p = 0.011), frontal association cortex (p = 0.024), and entorhinal cortex (p = 0.021),

while chronically-exposed males showed changes in ACD in the parabrachial area (p = 0.001),

endopiriform area (p = 0.001), and anterior commissure (p = 0.003).

Novel Object Preference

Male and female control groups displayed a preference for exploring novel objects over

familiar objects after a 5-minute inter-trial interval (ITI) as represented by a discrimination ratio

(DR) significantly greater than chance (male+placebo: DR = 0.66, p = 0.033 / female+placebo:

DR = 0.75, p = 0.22), as shown in Fig. 4. The female cannabis exposure group also displayed a


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significant preference for a novel object (female+cannabis: DR = 0.77, p = 0.002). Only males

exposed to chronic cannabis showed a no significant preference for a novel object when compared

to chance (Male+cannabis: DR = 0.605, p = 0.29). Although the NOP is a preference test, one

plausible interpretation would be that adolescent male mice cannot remember the familiar object

they were presented with 5 minutes earlier.

Discussion

The aim of this exploratory multimodal neuroimaging study was to address the preclinical

knowledge gaps as well as clinical limitations in the inhaled cannabis research space, particularly

involving neuroimaging, one of the only directly clinically translatable methods currently

available. As such, while we acknowledge that there are limitations to our studies (i.e. awake vs.

anesthetized imaging, using more cognitive tests etc.) we are hopeful that these methods and

resulting data will inform and also bridge the gap between the preclinical cannabis studies and the

clinical findings, while opening the door to further research.

The behavioral analysis points at a sex difference in novel object preference performance,

with males showing impairment compared to females exposed to inhaled cannabis, which in turn

begs the question: why are females seemingly protected against the effects of inhaled cannabis

compared males? Indeed, cognitive deficits (i.e. working memory, attention etc.) were expected

based on clinical studies, particularly if exposure was chronic and initiated in adolescence (for

review, see Volkow et al., 2016). Furthermore, a number of preclinical studies point at sex-

dependent differences in cognitive performance, with some data showing a detrimental effect on

males while others reporting the opposite, particularly when exposure is chronic and initiated in

adolescence. Given the potential differences in methodology, particularly surrounding the NOP

test, it is difficult to reconcile the current findings with those reported previously (for review, see


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O'Shea et al., 2006), although focus has been placed on differences in neurodevelopmental

trajectories between males and females, as well as interaction between steroid hormones and

cannabinoids.

Sex differences were observed in connectivity, volumetric analyses and

diffusion/anisotropy. Males and females chronically exposed to vaporized cannabis both showed

decreases in connectivity between brain regions implicated in reward (substantia nigra, VTA),

learning & memory (hippocampus, CA1/CA3), and sensory information relay (thalamus)

compared to the control groups. Despite these similarities, only males exposed to cannabis showed

a unique pattern of hypoconnectivity, specifically between key limbic areas (thalamus,

hippocampus) and the BNST, a center for the integration of limbic information and valence

monitoring. This pattern of hypoconnectivity in males may suggest a dysregulation in limbic

system information processing and relay to other regions in males, which could ultimately underlie

the cognitive effects observed in this group. These results are in part consistent with clinical

findings showing decreases (to a lesser extent) and increases in functional connectivity in response

to inhaled cannabis, and these changes were noted primarily in CB1-receptor-rich areas (Klumpers

et al., 2012). Similar findings were reported by Harding et al. (2012), whereby this reported

increased in connectivity was attributed to a likely compensatory role in mitigating cannabis-

related impairments in cognitive control or perceptual processes.

Although chronic cannabis exposure appears to result in a greater degree of functional

connectivity changes in males, females exposed to the same condition showed a larger number of

significant structural changes to distinct brain regions (17 ROIs) compared to males (5 ROIs).

Furthermore, volumetric changes in females include both decreases (locus coeruleus, VTA,

cerebellum) as well as increases in volume (olfactory tubercles, lateral dorsal thalamus, ACC),


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while males only showed decreases in volumes (central/medial amygdaloid areas, ambiguous area,

caudate putamen, superior colliculus). Although this remains to be tested, it is possible that these

changes could be an adaptive response to the treatment, considering that females did not show

cognitive impairment on the NOP test compared to males.

Data collected from DWI scans show that males and females share some changes in white

matter microarchitecture as shown by differences in ACD, or apparent diffusion coefficient.

Regions where ACD was significantly different from controls in both males and females include

superior colliculus, nucleus accumbens core, dorsal raphe, locus coeruleus, periaqueductal gray,

subiculum, dentate gyrus, insular rostral cortex, and ventral pallidum. Only females show distinct

changes in ACD in areas including the dorsal hippocampal commissure, orbital cortex, medial

geniculate, frontal association cortex, and entorhinal cortex, while only males showed changes in

the parabrachial area, endopiriform area, and anterior commissure. These findings suggest that

chronic exposure to inhaled cannabis has an effect on white matter microarchitecture, and these

effects are different between sexes. These effects support recent clinical data linking chronic

cannabis use to microarchitectural changes (Cookey et al., 2018).

Despite the neuroradiological differences yielded by these multi-modal assessments seem

to be sex-dependent, it would be premature to attempt to correlate the behavioral and imaging data,

or even attempt a link between our findings and the documented sex differences in CB1 receptor

distribution and estrous cycle-related receptor changes in receptor sensitivity (de Fonseca et al.,

1994), while Klumpers et al. (2012) report males showing better tolerability to THC effects

compared to females. To that end, follow-up studies should be prudently designed with this very

goal in mind (assessing the nature of these sex-differences using preclinical multi-modal

neuroimaging).


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Conflict of interest:

CFF has a financial interest in Animal Imaging Research, the company that makes the RF

electronics and holders for animal imaging.


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Figures & legends:

Figure 1 - Above are correlation matrices resting state BOLD functional connectivity comparing 140 brain

areas between placebo and marijuana groups for each gender. Chronic exposure to inhaled marijuana during

adolescence affects rs-fcMRI in both males and females. Labelled areas showing significant changes to BOLD

activation include: A - brainstem reticular activating system; B – anterior cerebellum; C – raphe/pons

connections to paracrachial/trigeminal; D – pons/lemniscal system; E – PAG/mesencephalic reticular area; F

– accumbens/prefrontal cortex; G – caudate putamen/ventral pallidum; H – substantia nigra/VTA

connections to midbrain; I – hypothalamus/thalamus; J – CA1/CA3 connections to thalamus; K – midline

thalamic areas/hypothalamus; L – hippocampus/midbrain; M – raphe negative connectivity with limbic

thalamus and hippocampus; N – hypothalamus/thalamus negative connectivity to BNST and temporal

cortex. Below are charts and 3-D activation maps showing areas with positive (yellow) and negative (blue)

connectivity to female area G (ventral pallidum) and male area M (raphe). Note the increase in negative

connectivity in males area M chronically exposed to vaporized marijuana compared to females which do not

share these hypoconnectivity changes.


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Figure 2. Above are probability maps heat maps for significant differences between placebo and marijuana

for females from voxel based morphology scans. Table 1 lists 17 affected female brain volumes in order of

significance, of which olfactory tubercles, VTA, and accumbens shell are of particular relevance to

interactions with the reward system. Table 2 lists 5 affected male brain volumes in order of significance, not

including any of the reward areas affected in females. Results show chronic exposure to inhaled marijuana

during adolescence affects both area- and sex-specific brain volumes in both males and females.


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Figure 3. Probability heat maps for significant differences in apparent diffusion coefficient (ACD) between

placebo and marijuana for each gender as measured by diffusion weighted imaging (DWI) with quantitative

anisotropy. These maps show chronic exposure to inhaled cannabis during adolescence affects while matter

microarchitecture in both males and females in sex-specific regions.

Figure 4. Novel Object Preference results of the test trial in male and female mice. Both male and female

control groups, as well as the female cannabis group showed investigation rations significantly greater than change, while male cannabis group’s investigation ration was not significantly different than chance.


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Neuroradiological and behavioral consequences of repeated ... · research. I’d like to sincerely thank my faculty mentor, Craig Ferris, for giving me the opportunity and guidance