Cannabidiol Modulates Hippocampal Dependent Learning and Alters the Accompanying Synaptic ...

101 Pages·2017·5.31 MB·English

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Cannabidiol Modulates Hippocampal Dependent Learning
and Alters the Accompanying Synaptic Plasticity in the
Ventral Hippocampus.

A Master’s Thesis

Presented to the Faculty of the

College of Science and Mathematics

Colorado State University-Pueblo
Pueblo, Colorado

In Partial Fulfillment
Of the Requirements for the Degree of

Master of Science in Biology

By

Connor Balkissoon

Colorado State University - Pueblo

(May 2018)
CERTIFICATE OF ACCEPTANCE

This Thesis Presented in Partial Fulfillment
of the Requirements for the Degree

Master of Science in Biology

By
Connor Balkissoon

Has Been Accepted
By the Graduate Faculty of the

College of Science and Mathematics

Colorado State University-Pueblo

APPROVAL OF THESIS COMMITTEE

Graduate Advisor (Dr. Jeffrey P. Smith) Date

Committee Member (Dr. Brian Vanden Heuvel) Date

Committee Member (Dr. Barbara Brett) Date

Graduate Director (Dr. Claire Ramos) Date
DEDICATION

Id like to dedicate this to my mother and father
who have always been there in supporting me
so that I could follow any passion I please.

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ACKNOWLEGDMENTS
I’d like to acknowledge my advisor Dr. Jeff Smith for giving me this
opportunity. Who has stuck with me and helped me develop skills that
stretch beyond biology and helped me grow as a person.

I’d also like to acknowledge my committee members, Dr. Brian Vanden
Heuvel and Dr. Barbra Brett, for dedicating their time and giving their
advice.

I’d also like to acknowledge the other members in our lab, Amy Uhernik,
Zack Montoya and Jose Vigil that have helped in countless ways throughout
this process. Explicitly I must thank Amy and Zach for doing the behavioral
portion of this work, this was not possible with out their help.

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TABLE OF CONTENTS
DEDICATION i
ACKNOWLEDGEMENTS ii
ABSTRACT iv
LIST OF FIGURES v
BACKGROUND AND SIGNIFICANCE
Medical marijuana as a proposed treatment for anxiety disorders. 1
Cannabidiol could be beneficial for treating PTSD. 5
Cannabidiol may target synaptic plasticity in the hippocampus and 8
amygdala to affect fear conditioning.
MATERIALS AND METHODS
Behavioral Testing 12
Histology 15
Imaging and selection of neurons 21
Morphological Analysis 25
Solutions 34
RESULTS
Behavioral outcomes of fear conditioning 36
Morphological outcomes post-fear conditioning. 45
DISCUSSION
Synaptic plasticity occurred after learning 51
CBD moderately effected synaptic plasticity 52
CBD’s effect on synaptic plasticity is helpful for explaining 56
behavioral outcomes.
Summary 59
LITERATURE CITED 61
APPENDICES
A: PUBLISHED ARTICLE 80
B: EXCEL DATA SHEETS 89
C: THESIS PRESENTATION SLIDES 91
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ABSTRACT
Cannabidiol (CBD) is thought to have therapeutic potential for treating
psychiatric conditions that affect cognitive aspects of learning and memory, including
anxiety and post-traumatic stress disorder (PTSD). Studies have shown that CBD
enhances extinction of fear memory when given after conditioning. This led us to
hypothesize that CBD might also enhance the acquisition of new fear memories.
Furthermore, PTSD has cognitive and reflexive components, so we employed trace and
delay fear conditioning which differ in their cognitive demands. Since changes in
dendritic spine density and spine lengths are indicators of synaptic plasticity, and
synaptic palsticity is thought to be a mechanism for learning and memory, we also
hypothesized that CBD may modulate the synaptic palsticity that accompanies the
formation of fear memories. We show that CBD-treated trace fear conditioned mice had
increased levels of freezing during conditioning, inhibited cue-dependent memory
extinction, slightly increased levels of freezing during an auditory-cued memory test and
increased contextual fear memory. Conversely, CBD-treated delay conditioned mice only
showed increased freezing levels during contextual conditioning. By measuring spine
density and spine length in the ventral hippocampus and amygdala, we show that CBD
modulated synaptic plasticity in the hippocampus while having minimal effects on
synaptic plasticity in the amygdala. Overall, the modulating effects of a single pre-
conditioning dose of CBD on memory and synaptic plasticity, demonstrate the need to
more fully characterize its basic effects on memory, suggest caution when using it
clinically as an anxiolytic, and point to a need for more research into its potential as a
therapeutic for treating memory-loss disorders.

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List of Figures

Figure 1: Mounting area set up. 16
Figure 2: Slicing area set up. 17
Figure 3: Trimming and alignment of impregnated brain. 18
Figure 4: Folder organization. 23
Figure 5: Lateral amygdala. 24
Figure 6: Hippocampal regions. 25
Figure 7: Spine and Length measurements. 29
Figure 8: Output of raw data measurements logged from MetaMorph. 30
Figure 9: Length of spines. 31
Figure 10: Partial master Excel sheet. 32
Figure 11: Example table that compiles data for each dendritic segment within 33
a brain region for a single neuron
Figure 12: Trace and Delay fear conditioning produced elevated freezing in 37
both vehicle and CBD treatment groups.
Figure 13: Animals treated with CBD just prior to trace-fear conditioning had 39
modulated fear memory.
Figure 14: Animals treated with CBD just prior to delay fear conditioning did 41
not effect on fear memory.
Figure 15: Context-dependent freezing was enhanced by CBD. 43
Figure 16: CBD treated animals showed an increased level of freezing during 44
conditioning.
Figure 17: CBD did not alter synaptic plasticity in the lateral amygdala. 46
Figure 18: CBD altered synaptic plasticity in the hippocampal CA1 region. 47
Figure 19: CBD altered synaptic plasticity in the hippocampal CA3 region. 49
Figure 20: CBD minimally effected synaptic morphology. 50

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Background & Significance
Medical marijuana as a proposed treatment for anxiety disorders
It’s estimated that the annual cost to society of anxiety disorders is 79 billion
dollars (Sidran Institute, 2016). Approximately 15.7 million people in the U.S. each year
will suffer from an anxiety disorder (Lépine, 2002). The economic cost of anxiety
disorders come not only in the form of treatment from medical professionals and
pharmaceuticals, but also reduced productivity, increased rates of unemployment and
elevated suicide risk (Blessing et al., 2015; Lépine, 2002). Post-traumatic stress disorder
(PTSD) is an anxiety disorder that can form after experiencing a life-threatening event
such as a natural disaster, mass violence, a car accident or combat. Although the vast
majority of people who experience traumatic events will exhibit PTSD-like symptoms
one week after the event, only approximately 10% will develop the disorder (Foa et al.,
1991; Breslau, 2009). Even though only a small subset of people develops the disorder,
approximately 8 million adults each year, and 8% of the U.S. population over their
lifetime, will have PTSD (“How common is PTSD?”, 2017). A report by the
congressional budget office found that the average cost of care to treat PTSD in Iraq war
veterans was eight thousand dollars per patient in the first year of treatment
(Congressional Budget Office, 2012). Over the second, third and fourth years of
treatment, the cost declined, but stabilized at four thousand dollars per year
(Congressional Budget Office, 2012). Notably, even though the first year of treatment
was the most expensive, much of expenses come in the continual treatment in the
following years. This illustrates that there’s currently no effective treatment for curing
PTSD.

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Currently, selective serotonin reuptake inhibitors (SSRI’s) are the only FDA
approved medications for the treatment of PTSD and considered “the first-line
pharmacological treatment for PTSD” (Berger et al., 2009). SSRI’s have a 60% overall
response rate and a 20% to 30% rate of achieving complete remission in PTSD patients
(Berger et al., 2009). Lack of complete remission and deficient response rates not only
keep costs high, but also leads patients to seek other treatment options. Additional
pharmacological treatment options include benzodiazepines, opiates, selective-
norepinephrine reuptake inhibitors, partial 5-hydroxytryptamine receptor agonists and
medical marijuana, all of which can have adverse side effects, can be addictive, or can
lack efficacy (Bonn-Miller & Rousseau, 2017; Blessing et al., 2015; Joy et al., 1999).
The efficacy of medical marijuana for anxiety disorders like PTSD is
inconclusive. This is in part due to the contradictory effects that cannabis can have based
on dose and inborn genetic variability (Harvard Medical School, 2010). Medical
marijuana is legal in 29 states; however, it’s classified by the Veteran’s Association as a
substance use disorder (Bonn-Miller & Rousseau, 2017; ProCon.org, 2017). Use of
marijuana by veterans has been increasing steadily over the past ten years and is currently
the most common substance use disorder (Bonn-Miller & Rousseau, 2017). Direct data
about the efficacy of medical marijuana and its therapeutic value specifically for PTSD is
limited; although there are anecdotal reports from veterans who say it’s helpful for
relieving symptoms (Bonn-Miller & Rousseau, 2017). In 2014, a study backed this by
showing that 75% of patients with PTSD that used medicinal marijuana had a significant
reduction in symptoms; however, this study did not use the placebo controls that are
necessary to determine cannabis’ efficacy (Greer, Grob & Halberstadt, 2014). Most
studies on the health effects of cannabis use focus on subjects that started using during

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adolescence or early adulthood and have used at very high potencies (Degenhardt & Hall,
2008; Joy et al., 1999). From this data, chronic recreational use of cannabis can lead to
chronic bronchitis, exacerbation of cardiovascular disease, impairment of short-term
memory, psychosis, and impairment of cognitive ability (Bonn-Miller & Rousseau, 2017;
Degenhardt & Hall, 2008). Recently, emergency rooms have started to see more cases of
cannabinoid hyperemesis syndrome which arises from chronic use at high doses and
involves cyclic episodes of nausea and vomiting (Galli et al., 2011). There is also a small
possibility of developing a dependency that leads to short-lived withdrawal symptoms,
however, this risk is less than other prescribed anti-anxiety medications (Benson et al.,
1999).
Marijuana is estimated to contain 545 compounds; of those compounds, 104 are
phytocannabinoids (Pertwee, 2014; ElSohly & Slade, 2005). Phytocannabinoids are
unique to cannabis and refers to eleven main compounds, which are all similar in having
a twenty-one carbon terpenophenolic skeleton, and their derivatives (Pertwee, 2014;
ElSohly & Slade, 2010). There are several strains of cannabis, all of which vary in the
composition of phytocannabinoids (McLaren et al., 2008). Moreover, the composition of
phytocannabinoids within the same strain can vary by more than 25% (Hazekamp &
Fishedick, 2012). Though only a few phytocannabinoids are known to alter synaptic
transmission, the effects on synaptic transmission and the neuronal areas affected can be
vastly different dependent on the phytocannabinoid (Szabo, 2014). The inherent variance
in phytocannabinoid concentrations between, and even within strains, is likely to be part
of the reason for conflicting reports as to the therapeutic value of medicinal marijuana
(McLaren et al, 2008).

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