Cannabinoid Type 1 Receptor Expression, Synaptic Function, and Perturbation

CANNABINOID TYPE 1 RECEPTOR EXPRESSION, SYNAPTIC FUNCTION, AND PERTURBATION

WITHIN THE DEVELOPING CEREBELLAR CORTEX

JESSE LEE BARNES

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Program in Neuroscience

JULY 2019

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of JESSE LEE

BARNES find it satisfactory and recommend that it be accepted.

David J. Rossi, Ph.D., Chair

James Peters, Ph.D.

Ryan McLaughlin, Ph.D.

Suzanne Appleyard, Ph.D.

Gary Wayman, Ph.D.

ACKNOWLEDGEMENT

I would like to acknowledge my mentor David Rossi for helping me grow as a critical thinker and aiding in my understanding of experimental design. Thank you Claudia Mohr for teaching me the patch-clamp technique in slice when I first joined the lab. Of course Hiroko Shiina, my fellow lab mate, offered so much support and made lab work enjoyable, as well as helped with some of my experiments. It has also been a joy to watch Chloe and Halle develop as junior graduate students.

Also I’d like to thank my committee (Dr. James “Jimmy” H. Peters, Suzanne A. Appleyard, Ryan J.

McLaughlin, and Gary A. Wayman) for the advice and guidance in all things both in and out of the lab. The vivarium staff also aided in making this research possible, and the departmental staff contributed greatly by all the help they’ve offered.

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CANNABINOID TYPE 1 RECEPTOR EXPRESSION, SYNAPTIC FUNCTION, AND PERTURBATION

WITHIN THE DEVELOPING CEREBELLAR CORTEX

Abstract

by Jesse Lee Barnes, Ph.D. Washington State University July 2019

Chair: David J. Rossi

The endocannabinoid receptor 1 (CB1R) plays a neuromodulatory role throughout the central nervous system. A large proportion of CB1Rs are concentrated within the cerebellar cortex, a brain region known to control motor coordination and motor learning, but has been gaining recognition for cognitive involvement. A critical period of cerebellar development occurs during the third trimester of pregnancy in humans and the early postnatal period in rodents, a period involving neuronal proliferation, migration, and synaptogenesis. The endocannabinoid system has been implicated in playing a role in numerous neurodevelopmental processes. However, we lack a clear understanding of whether the endocannabinoid system is established during cerebellar growth, or whether cannabinoid receptor activation modulates trans-cellular signaling and other processes known to modulate neurodevelopment. Using a neurodevelopmental rodent model equivalent to third trimester human fetal tissue, we observed prominent and temporally unique immunohistochemical CB1R expression within all layers of the cerebellar cortex. Using patch-clamp electrophysiology, we characterized how CB1Rs modulate synaptic transmission. We observed CB1R actions at synapses onto granule cells, Golgi cells, and

ii stellate/basket cells, but not Purkinje cells. This is contrary to mature Purkinje cell terminals, which exhibit a prominent reduction in vesicle release probability in response to cannabinoids. It is well established that granule cell migration rate is influenced by ambient glutamate, but it is currently unknown whether parallel fibers are the source of this glutamate, or whether CB1Rs can modulate glutamate levels. We therefore determined mossy fiber CB1R activation attenuates downstream parallel fiber glutamate release onto migrating granule cells, demonstrating that cannabinoids may mediate migration by controlling parallel fiber glutamate release. To explore this last point, we exposed rat pups to an exogenous cannabinoid and later subjected them to an accelerated rotarod task during adolescence. Cannabinoid-exposed rats showed deficiencies in learning the task, suggesting altered cerebellar development. The results of this dissertation provide evidence that CB1R expression within the developing cerebellar cortex uniquely modulates synaptic function during a critical period of cerebellar development, and exogenous

CB1R activation may have developmentally exclusive behavioral implications. These implications may ultimately aid in shaping guidelines for cannabis use in pregnant women.

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TABLE OF CONTENTS Page ACKNOWLEDGEMENT ...... iii

ABSTRACT ...... ii

TABLE OF CONTENTS...... iv

LIST OF FIGURES ...... ix

LIST OF TABLES...... xi

ABBREVIATIONS...... xii

CHAPTER 1 ...... 1

1.1 Overview ...... 1

1.2 Cerebellum ...... 3

1.2.1 Overview of cerebellum ...... 3

1.2.2 Circuitry of the cerebellar cortex ...... 4

FIGURE 1.1 ...... 9

1.3 Cerebellar Function ...... 9

1.4 Cerebellar development ...... 10

1.4.1 Granule cells ...... 11

FIGURE 1.2 ...... 13

1.4.2 Purkinje cells ...... 14

1.4.3 Cerebellar Cortex Inputs: Mossy fibers and climbing fibers ...... 15

1.4.4 Rodent age equivalent of third trimester human fetuses ...... 16

1.5 Cannabinoid receptors and their ligands ...... 17

1.5.1 Cannabinoid Receptors ...... 17

1.5.2 Cannabinoid receptor CNS distribution ...... 19

1.5.3 Mechanisms of cannabinoid receptors...... 20

1.5.4 Cannabinoids ...... 22

1.5.5 Role of endocannabinoid system in synaptic modulation ...... 24

1.5.6 Role of endocannabinoid system in neurodevelopment ...... 26

1.6 Endocannabinoid System within the Cerebellum ...... 30

1.6.1 Cannabinoids in adult cerebellar cortex ...... 30

1.6.2 Cannabinoids in developing cerebellar cortex ...... 32

1.7 Effects of Neurodevelopmental Cannabinoid Exposure ...... 34

1.7.1 Motor Function ...... 35

1.7.2 Non-motor Behavioral Effects...... 37

1.8 Neurocognitive Disorders Associated with Cerebellar Dysfunction...... 39

1.8.1 Autism Spectrum disorder ...... 40

1.9 Therapeutic Effects of Exogenous Cannabinoids ...... 41

1.10 Summary/Discussion ...... 42

CHAPTER 2 ...... 45

ABSTRACT ...... 45

INTRODUCTION ...... 46

MATERIALS AND METHODS ...... 48

RESULTS ...... 53

FIGURE 2.1 ...... 71

FIGURE 2.2 ...... 72

FIGURE 2.3 ...... 74

FIGURE 2.4 ...... 75

FIGURE 2.5 ...... 77

FIGURE 2.6 ...... 79

FIGURE 2.7 ...... 81

FIGURE 2.8 ...... 82

FIGURE 2.9 ...... 84

FIGURE 2.10 ...... 85

FIGURE 2.11 ...... 87

FIGURE 2.12 ...... 88

FIGURE 2.13 ...... 89

FIGURE 2.14 ...... 90

FIGURE 2.15 ...... 92

FIGURE 2.16 ...... 94

FIGURE 2.17 ...... 95

FIGURE 2.18 ...... 96

FIGURE 2.19 ...... 97

FIGURE 2.20 ...... 99

FIGURE 2.21 ...... 101

TABLE 2.1 ...... 103

DISCUSSION ...... 104

CHAPTER 3 ...... 116

ABSTRACT ...... 116

INTRODUCTION ...... 117

METHODS ...... 120

RESULTS ...... 125

FIGURE 3.1 ...... 127

FIGURE 3.2 ...... 128

DISCUSSION ...... 130

CHAPTER 4 ...... 136

ABSTRACT ...... 136

INTRODUCTION ...... 137

METHODS ...... 140

RESULTS ...... 144

FIGURE 4.1 ...... 152

FIGURE 4.2 ...... 153

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FIGURE 4.3 ...... 154

FIGURE 4.4 ...... 155

FIGURE 4.5 ...... 156

FIGURE 4.6 ...... 157

FIGURE 4.7 ...... 158

TABLE 4.1 ...... 159

TABLE 4.2 ...... 160

TABLE 4.3 ...... 161

DISCUSSION ...... 162

CHAPTER 5 ...... 170

Overview...... 170

CB1R localization and effects on synaptic transmission ...... 171

Cerebellar network effects of CB1R activation ...... 174

Exogenous cannabinoid influence on cerebellar development ...... 176

Future directions ...... 178

Concluding remarks ...... 180

REFERENCES ...... 183

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LIST OF FIGURES

FIGURE 1.1 ...... 9

FIGURE 1.2 ...... 13

FIGURE 2.1 ...... 71

FIGURE 2.2 ...... 72

FIGURE 2.3 ...... 74

FIGURE 2.4 ...... 75

FIGURE 2.5 ...... 77

FIGURE 2.6 ...... 79

FIGURE 2.7 ...... 81

FIGURE 2.8 ...... 82

FIGURE 2.9 ...... 84

FIGURE 2.10...... 85

FIGURE 2.11...... 87

FIGURE 2.12...... 88

FIGURE 2.13...... 89

FIGURE 2.14...... 90

FIGURE 2.15...... 92

FIGURE 2.16...... 94

FIGURE 2.17...... 95

FIGURE 2.18...... 96

FIGURE 2.19...... 97

FIGURE 2.20...... 99

FIGURE 2.21...... 101

FIGURE 3.1 ...... 127

FIGURE 3.2 ...... 128

FIGURE 4.1 ...... 152

FIGURE 4.2 ...... 153

FIGURE 4.3 ...... 154

FIGURE 4.4 ...... 155

FIGURE 4.5 ...... 156

FIGURE 4.6 ...... 157

FIGURE 4.7 ...... 158

LIST OF TABLES

TABLE 2.1 ...... 103

TABLE 4.1 ...... 159

TABLE 4.2 ...... 160

TABLE 4.3 ...... 161

ABBREVIATIONS aCSF Artificial cerebral spinal fluid

CB1R Cannabinoid Receptor Type 1

CF Climbing Fiber

EGL External Granule Layer

GBZ Gabazine

GC Granule Cell

GCL Granule Cell Layer

GL Granule Layer (Same as GCL)

MF Mossy Fiber

ML Molecular Layer

PC Purkinje Cell

PCL Purkinje Cell Layer

PL Purkinje Layer (Same as PCL)

SR SR141716A (CB1R inverse agonist)

TTX Tetrodotoxin

WIN WIN 55,212-2 (CB1R agonist; CB2R agonist)

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CHAPTER 1

Introduction

1.1 Overview

Humans have been consuming Cannabis sativa L. (“cannabis”, or “marijuana”) for its psychoactive and medicinal properties for thousands of years1,2. Globally, nearly 119-224 million people—2.6 to 5 percent of the adult population—currently consume cannabis on an annual basis3. In the United States alone, 4.8% of adolescents between the ages of 12 and 17 tried cannabis for the first time within the past 12 months4. Cannabis use has been rapidly increasing, with a resurging interest in the western world to legalize its use for both recreational and medicinal purposes.

The most common age group consuming cannabis within the United States is 18-25 years old4, an age when humans are undergoing the last stages of neurodevelopment. In addition, roughly 3% of pregnant mothers consume cannabis during the third trimester4 of pregnancy.

Although a relatively small percentage, this consists of roughly 130,000 pregnancies within the

United States in 2013. This is significant because a large proportion of the cerebellum develops during the third trimester of pregnancy until about 9 months post-utero in humans5. The prevalence of cannabis consumption may also depend on the stage of pregnancy, with pregnant women reporting cannabis consumption during the first (13.1%), second (5.1%), and third (5.0%) trimesters in a human longitudinal study6. There have been a number of observational studies

attempting to determine the impact of cannabis consumption on fetal and adolescent development, but results of the current literature leaves much to me learned.

Despite the popularity of cannabis for such an extended part of human history, scientists have only recently discovered how cannabis exerts its effects on the brain. The initial isolation and discovery of the phytocannabinoid (−)-trans-Δ9-tetrahydrocannabinol (THC) in the 1960’s7 has spearheaded the push for more cannabinoid research, leading the way to the discovery of multiple cannabinoid receptors, endogenously produced cannabinoids within the central and peripheral nervous systems, and the enzymes associated with producing and degrading them.

These initial discoveries encompass the study of the endocannabinoid system as we know it today.

There is still much to learn regarding how the endocannabinoid system develops throughout the central nervous system, and even less well understood is the neurodevelopmental effects of cannabinoid receptor activation. This is significant given the large population of pregnant women and adolescents using high concentration cannabis4. More recent research has attempted to characterize how the endocannabinoid system impacts neuronal development, and has found that the endocannabinoid system can modulate cell proliferation, migration, and dendritic arborization, among other modulatory roles8. The organized structure, the well-defined circuitry, and the convenient time in which the cerebellar cortex undergoes the majority of its development makes it a model system for studying whether cannabinoid receptors are uniquely expressed during a time period equivalent to third trimester pregnancy and early adolescence in humans, and the impact they may have on neurodevelopment.

The potential use of cannabinoids for medicinal purposes has been intensely studied recently. Cannabinoids contain many therapeutic qualities including anti-nausea properties, slowing progression of glaucoma, and have the potential to alleviate/prevent epileptic seizures, among other health benefits9,10. Furthermore some of these medicinal properties may be developmentally relevant, such as the assuagement of epileptic seizures in children, providing an alternative in treatment-resistant individuals. Therefore, research on both the effects of exogenous cannabinoids on the developing nervous system and their promising therapeutic implications has great significance due to current acceptance and legalization of cannabis.

Due to the association between cannabis consumption during a time of massive cerebellar growth and the discovery of endocannabinoid-mediated neurodevelopment, there is interest in determining how the endocannabinoid system may impact the developing cerebellum. Building on the limited scope of previous literature examining cannabinoid effects in mature cerebellar tissue, we hope to develop a better understanding of how cannabinoid receptors can impact developing cerebellar synaptic function. This will ultimately enable a better understanding of the role for the endocannabinoid system in development and the impact of its perturbation.

1.2 Cerebellum

1.2.1 Overview of cerebellum

The cerebellum controls neuronal processing underlying sensorimotor and vestibular motor control11 . Broadly, the cerebellum is involved in the maintenance of balance and posture,

coordination of voluntary movements, motor learning, and certain cognitive functions such as language12–14. Early cerebellar lesion studies have found deficits in coordination and gait, which are obvious behavioral phenotypes that initially drove the narrow perception of the cerebellum primarily functioning as a planner and coordinator of motor movement. When we consider gross cerebellar anatomy, there are specific subregions of the cerebellum. This includes: 1) the vestibulocerebellum, which is involved in vestibular reflexes and postural maintenance, 2) the spinocerebellum (including vermis), which is involved in integration of sensory input with motor commands to produce adaptive motor coordination, and 3) the cerebrocerebellum, involved in planning and timing of movement; as well as cognitive functions12,15.

In addition to many of the aforementioned motor components of cerebellar processing, there has been recent appreciation for a cerebellar role in cognition, emotion, and linguistic skills14. In fact, Cerebellar Cognitive Affective Syndrome (CCAS) has been coined to refer to behavioral deficiencies such as executive, visual spatial, linguistic impairments, and affective dysregulation that result from cerebellar lesions16. These observations implicating the cerebellum in aspects of cognition reveals a new avenue of research into additional functional roles, as well as understanding cognitive/psychiatric disorders12,15,17,18.

1.2.2 Circuitry of the cerebellar cortex

The cerebellar cortex is comprised of a highly organized neuronal circuit (Fig. 1, for schematic diagram of the circuit) with two excitatory glutamatergic inputs, climbing fibers and mossy fibers; climbing fibers synapse directly onto Purkinje cells, whereas mossy fibers synapse onto granule

cells upstream within the cerebellar cortex. Climbing fiber input originates from the inferior olivary nucleus within the medulla and passes through the pons and inferior cerebellar peduncles, which then synapse onto Purkinje cells and neurons within the deep cerebellar nuclei19. Climbing fiber activation may serve as a motor error signal to Purkinje cells, activating

Purkinje cells and disinhibiting deep cerebellar nuclei, which is an important motor timing signal but may also encode sensory processing and cognitive tasks13,20. Mossy fiber input originates from the pontocerebellar pathway, with axons passing through the middle and inferior cerebellar peduncles and synapsing with deep cerebellar nuclei and granule cells within the cerebellar cortex19. Purkinje cells act as the sole inhibitory GABAergic output from the cerebellar cortex, sending efferents to nuclei within the deep cerebellum. The majority of output fibers from the cerebellum originate from deep cerebellar nuclei, which then send projections out to the inferior olive and descending motor systems.

The mature rodent cerebellar cortex is comprised of 10 lobules, with each lobule composed of a number of segregated layers. White matter makes up the innermost portion of each lobule, which includes the axons of afferent mossy fibers and climbing fibers, and efferent

Purkinje cell axons. The granule cell layer (GCL) is the innermost layer comprised primarily of glutamatergic granule cell bodies, but is also comprised of Golgi cell GABAergic interneurons. The

Purkinje cell layer (PCL) is comprised of a single row of GABAergic Purkinje cells, and the outermost layer, the molecular layer (ML), is made up of Purkinje dendritic trees, glutamatergic granule cell bifurcated axons called parallel fibers, and GABAergic interneurons stellate and basket cells.

Granule Cells.

Granule cells are small, electrically compact, glutamatergic neurons that make up the majority of neurons within the GCL. They have a small number of dendrites (4-5)21 and receive excitatory glutamatergic input from mossy fibers and unipolar brush cells, and inhibitory GABAergic input from Golgi cells. Granule cell axons, called parallel fibers, extend up into the molecular layer, where they bifurcate and make excitatory glutamatergic synaptic connections with GABAergic

Purkinje cells, basket cells, stellate cells, and Golgi cells. Granule cells comprise a portion of one of the primary glutamatergic circuits onto Purkinje cells, and due to their high numbers compared to mossy fiber afferents, act to expand glutamatergic inputs onto Purkinje cells.

Purkinje cells.

GABAergic Purkinje cells are the sole output of the cerebellar cortex and receive excitatory glutamatergic input from two sources. First, each Purkinje cell receives a single excitatory input from climbing fiber afferents which originate within the olivary complex. Despite only one climbing fiber afferent innervating a single Purkinje cell, they can form up to 500 individual synapses, resulting in a powerful all-or-nothing excitatory input, and often act in response to a motor error. Second, Purkinje cells receive the majority of their excitatory input from granule cell axons which exist as bifurcated parallel fibers within the molecular layer of the cerebellar cortex.

Purkinje cells are complex integrators of signals and their cell firing dynamics can change, generating simple spikes in response to parallel fiber input and complex spikes in response to climbing fiber input22. Climbing fibers form many synaptic connections with Purkinje cells, so that their initiation of complex spikes in Purkinje cells raises enough calcium to transiently silence

parallel fiber-initiated simple spike firing, and induce long term plasticity23. This can also silence

Purkinje cell spontaneous firing24. Purkinje cells also receive inhibitory GABAergic inputs from stellate and basket cell interneurons. Although both neuronal types exist within the molecular layer, their axons synapse onto different parts of Purkinje cells: stellate cell synaptic terminals are canonical, forming synapses with Purkinje dendrites, whereas basket cell axons encapsulate the Purkinje cell axon hillock. Basket cell axons provide a powerful means of synaptic inhibition by more proximally mediating channel conductance near the site of signal propagation19. Once a

Purkinje cell action potential is initiated, its projections exit through the white matter and synapse onto deep cerebellar nuclei25.

GABAergic interneurons.

There are at least three primary GABAergic interneurons within the cerebellar cortex: one, the

Golgi cell, resides within the granule cell layer, whereas stellate and basket cells are located within the molecular layer. Golgi cells receive excitatory glutamatergic input from granule cell parallel fibers, and their only likely source of GABAergic inhibition is from other Golgi cells26. It has been traditionally believed that Golgi cells are synaptically contacted by basket cells and stellate cells due to Golgi cell dendrites projecting into the molecular layer However, recent evidence has concluded otherwise. In a 2011 study, mossy fibers were optogenetically stimulated while simultaneously recording from Purkinje cells and Golgi cells. If the latency of inhibition was the same, it would suggest molecular layer interneurons inhibit both Purkinje cells and Golgi cells.

The onset of inhibition onto Purkinje cells and Golgi cells differed, suggesting a different circuitry pathway. Additionally, simultaneous recordings of granule cells and Golgi cells during mossy fiber

stimulation shows they receive inhibitory input at approximately the same time, which is consistent with both cell types receiving inhibition from the same source26. Golgi cells form

GABAergic synaptic connections with granule cells, thus granule cell parallel fiber activation of

Golgi cells enables Golgi cells to reduce granule cell excitability. Similar to Purkinje and Golgi cells, stellate and basket cells receive excitatory glutamatergic input from parallel fibers, and receive

GABAergic input from fellow stellate and basket cells19. As GABAergic interneurons synapse onto

Purkinje cells, their primary role is to reduce Purkinje cell excitability.

FIGURE 1.1. Circuitry within the cerebellar cortex. The two inputs into the cerebellar cortex are glutamatergic mossy fibers and climbing fibers. The sole output of the cerebellar cortex are Purkinje GABAergic efferents, which project to deep cerebellar nuclei. The schematic depicted here represents the mature cerebellar cortex, but the same circuitry can be applied for developing tissue.

1.3 Cerebellar Function

The cerebellum has classically been considered the primary mediator of fine motor control and proprioception since motor ataxia represents one of the most clear behavioral deficits following cerebellar damage11. More broad behavioral roles include aspects of motor learning, control of voluntary limb movements, timing, and sensorimotor synchronization, which the cerebellum acts to fine tune through descending motor commands27–29. Part of these roles can be attributable to

input from a variety of brain regions, such as the prefrontal cortex, the motor cortex, and the sensory cortex16,30.

Traditionally, the cerebellum has been thought to control little more than fine motor functions. However, the role of the cerebellum has recently been considered to include more cognitive roles such as modulating emotion, attention, and memory31. Some of the first observations linking the cerebellum to cognitive and emotional affect occurred when there were noticeable cognitive behavioral deficiencies in patients with cerebellar lesions. These included executive function impairments in planning, set-shifting, verbal fluency, abstract reasoning, and working memory31. In addition, focal cerebellar injuries lead to specific cognitive deficits in reading and verbal working memory32. It was also recently found that different regions of the cerebellum become more active during a working memory task than during a motor task33³³ which, when all taken together, suggests the cerebellum is involved in a range of behavioral tasks beyond the scope of motor control.

1.4 Cerebellar development

The cerebellum initially develops through the migration of cell precursors from the rhombencephalon and eventually composes part of the metencephalon and rhombic lip. This primary germinal zone comprises the majority of neuronal precursors that eventually comprise the cerebellar cortex, while some of these precursors form a secondary germinal zone, the external granule layer (EGL), which will eventually become granule cells34. The cerebellar cortex has the same general circuitry structure as mature cortex, with the addition of the EGL, and undergoes the majority of its development during the third trimester of pregnancy in humans.

The development of cerebellar circuitry, although undergoing the vast majority during the third trimester, begins to mature after first two years postnatally35,36. Many neuronal circuits are overabundant during mammalian neurodevelopment, which serves as a refinement period when excessive synapses and dendritic processes are eliminated in favor of stronger, functionally ideal synapses37. Specifically to the cerebellum, granule cells within the cerebellar cortex undergo massive cell proliferation and migration, while all cells undergo dendritic proliferation19. This section focuses on neuronal development of the primary neurons of interest within the cerebellar cortex.

1.4.1 Granule cells

Development of the granule cell layer (GCL) begins when immature granule cell precursors begin to proliferate within the external granule layer38. Following proliferation, they tangentially migrating along the EGL, then turning 90° to radially migrate through the molecular layer (ML) along Bergman glial fibers39. Tangential granule cell migration through the EGL and ML switches to radial migration as soon as granule cells reach the GCL. At this phase of migration, granule cells are guided by a leading process that differentiates into a dendrite before reaching their post- migratory destination within the granule cell layer39.

Premature granule cells within the EGL rely in part on paracrine glutamatergic signaling40,41 and subsequent activation of NMDA receptors40,42,43 to drive their migration down

Bergman glial fibers. NR2B subunit-containing receptors are primarily expressed on immature granule cells during this early developmental phase, but are gradually replaced with NR2A and

NR2C-containing NMDA receptors, and overexpressing NR2B through mutant mouse lines increases granule cell migration44. However, there appears to be little spontaneous NMDA receptor activity in the premigratory zone of the EGL, whereas high levels of NMDA receptor activity is reported in the GCL45. This may indicate an increasing reliance on glutamate-mediated signaling when migrating granule cells near their final destination. Additionally, granule cells may also rely on activation of voltage-gated Ca2+ channels (VGCCs) to stimulate migration. Granule cells begin to primarily express N-type Ca2+ channels as they reach the bottom of the EGL, and selective N-type specific Ca2+ channel blockade slows ML migration rate. Inhibition of L- and T- type channels has no effect on migration rate46. Also, reducing Ca2+ influx by reducing extracellular Ca2+ or blocking NMDA receptors decreases the frequency and size of intracellular

Ca2+ fluctuations, which correlate with reduced neuronal migration47. This migration rate, along with the concurrent thickening of the molecular layer, increases with age, 48,49. This rate increase is presumably driven by the need for migrating granule cells to travel greater distances in a similar amount of time as the tissue thickens. Other than a role of glutamate in migration, there may be an internal granule cell migratory drive. In isolated granule cell cultures with an absence of normal external cues, migrating granule cells still display an intrinsic level of migration50. There are clearly multiple factors driving granule cell migration.

As granule cells migrate their axons extend into the molecular layer, bifurcate, and synapse onto a number of cell types, including Purkinje cells and inhibitory ML interneurons. The molecular layer grows larger with the arrival of more granule cell parallel fibers and the proliferation of Purkinje dendritic trees19, where there eventually forms a massive number of granule to Purkinje synaptic connections at 175,000 synapses per dendritic tree51. The sheer

number of parallel fiber -> Purkinje cell synaptic connections allows for Purkinje cells to receive a large range of synaptic inputs.

Post-migratory granule cells undergo dendritic extension and synaptogenesis; they must extend dendritic neurites in an attempt to form stable synaptic connections and pruning21,52–54.

There appears to be a relatively narrow developmental window of dendritic pruning, where granule cells have roughly 8-9 dendrites five days before maturation before being pruned back to an average of 4 dendrites two days before maturation21. Each granule cell dendrite forms a claw at its terminus, which is the site of synaptic contact between glutamatergic mossy fibers and

GABAergic Golgi cells (known as glomeruli; Fig. 2).

FIGURE 1.2. A depiction of a synaptic glomerulus within the granule cell layer of the cerebellar cortex. The glomeruli found within the granule cell layer is the point of synaptic contact between mossy fiber and Golgi terminals with granule cell dendrites. They are typically found in areas void of granule and Golgi cell nuclei. Red: glutamatergic mossy fiber axons; green: GABAergic Golgi cell axons; blue: granule cell dendrites.

In parallel with developmental processes such as migration and synaptogenesis, granule cell excitability may also be in flux. The reversal potential for chloride is different than mature

gradients, where GABAA receptors function to depolarize granule cells and increasing their excitability55. Compared with extracellular chloride levels, there is a higher concentration of intracellular chloride within neurons, which is counter to more developed granule cells. This is thought to be driven by the expression ratio of two chloride co-transporters: the Na+-K+-2Cl- co- transporter (NKCC1), and the K+-Cl- co-transporter (KCC2). NKCC1 transports chloride into the cell, and KCC2 transports chloride out56. NKCC1 expression is established before KCC2, so what defines the shift from depolarizing GABA to hyperpolarizing GABA is the eventual expression of

KCC2 This drives more chloride out of the cell and establishes the hyperpolarizing gradient. The chloride reversal potential appears to transition from nearly -40mV at PND 0, to -60mV at PND 7, where GABAAR activation begins to become canonically hyperpolarizing56. Functional studies have found increased calcium signaling in both cerebellar granule cells (PND 3-5)57 and Purkinje cells (PND 2-22)58 following application of a GABA agonist, suggesting depolarizing actions of

GABARs within the cerebellum. These data reveal that both glutamatergic and GABAergic neurotransmission onto granule cells may both be excitatory during an early developmental age.

1.4.2 Purkinje cells

Purkinje cells establish their location within the cerebellar cortex at birth, and develop their classic monolayer arrangement at postnatal (PND) 4-5 in mice59. Similar to granule cells, proper

Purkinje cell dendritic arborization may partially rely on glutamatergic signaling—Purkinje cell dendrites fail to develop normally in mice that have experiences massive granule cell elimination60. Granule cell parallel fiber glutamate release has been found to activate Purkinje

NMDA receptors, and subsequent intracellular Ca2+ increase and electrical activation of these aids in dendritic branching61. Additionally, Purkinje cell spine formation is severely stunted in

GluRδ2-deficient mice62, which further suggests a glutamatergic role in neuronal developmental plasticity.

1.4.3 Cerebellar Cortex Inputs: Mossy fibers and climbing fibers

Immature mossy fibers first migrate to the granule cell layer and reach a primary growth stage at

PND 6 in the rat, where mossy fiber rosettes undergo rapid enlargement63,64. Once granule cells begin to migrate to the granule cell layer, their dendrites begin to form immature asymmetrical synapses with the mossy fiber swelling before finally forming mature synaptic contacts.

Interestingly, mossy fibers attempt synaptic contacts with Purkinje cells early in development but

Purkinje cell-derived trophic factor BMP4 repels mossy fiber growth. This leads to their synapse elimination and allows for proper synaptic contacts with granule cells65.

Climbing fiber axons, the excitatory glutamatergic innervation which synapse directly onto Purkinje cells, originate from the inferior olive (olivary complex), where multiple climbing fibers initially establish glutamatergic synaptic connections with a single Purkinje cell. Redundant climbing fibers are generally eliminated as a single climbing fiber is strengthened relative to others, forming a one-to-one climbing fiber to Purkinje ratio37. Climbing fibers form multiple synapses onto single Purkinje cells during early rodent development (with 3-4 fibers by PND 5) with a high level of climbing fiber synchronization, but this leads to reduced synchronicity of

Purkinje population activity as climbing fiber synapses begin to prune. This is thought to be crucial

for the appropriate maturation of Purkinje cell populations66. Glutamatergic signaling is important for climbing fiber development as well, as drug-induced GluRδ2 ablation in mature

Purkinje cells leads to additional distal climbing fiber innervation of neighboring Purkinje cell dendrites, and fails to develop proper mono-innervation of Purkinje cells67.

1.4.4 Rodent age equivalent of third trimester human fetuses

Many rodent studies that attempt to model human neurodevelopment are often designed in a way that often miss the most critical period of cerebellar growth. For example, previous studies attempting to examine fetal effects of exogenous cannabinoids on cerebellar development have exposed pregnant rats to the CB1R agonist WIN 55,212-2 as a way to translate effects to maternal consumption68. However, when comparing brain growth, myelination, and cholesterol levels between human and rodent brains, postnatal days (PND) 1-10 appear to be developmentally equivalent to third trimester fetuses in humans69. The timetable for cerebellar development is fixed for each species, but varies between species. For example, and specifically within the cerebellum, cerebellar granule cell migration timeframes between humans and rodents are quite different. In humans, the eventual completion of granule cell migration occurs between 10 weeks into gestation until 2 months following birth, and the external granule layer disappears after 18 months70,71. In rodents, granule cells finish migrating out of the external granule layer by the third postnatal week69,72. It is now established that the rodent equivalent third trimester neurodevelopmental period occurs two postnatal weeks after birth, when the cerebellum is undergoing most its most pronounced developmental changes5. This makes investigating

development of the third trimester in rodents more experimentally simplistic, and provides rationale for performing experimental manipulations during this time period.

1.5 Cannabinoid receptors and their ligands

1.5.1 Cannabinoid Receptors

So far there are known to be two primary cannabinoid receptors that bind to cannabinoid ligands: cannabinoid receptor 1 (CB1R), first discovered in 199073, and cannabinoid receptor 2 (CB2R), discovered shortly after in 199374. CB1/2Rs are seven-transmembrane domain GPCRs, coupled

74,75 to Gi/Goα heterotrimeric G proteins, with CB2Rs exhibiting 44% homology with CB1Rs . The amino acid sequence of CB1Rs is highly conserved, exhibiting 97-99% conservation across a variety of species such as rats, mice, and humans76, whereas CB2Rs have 82% sequence identity between mice and humans77. In mature tissue, CB1Rs dominate cannabinoid receptor expression within the central nervous system, while CB2Rs are more widely expressed in the peripheral nervous system or within CNS immune cells. However, there is an increasing amount of research suggesting more ubiquitous expression of these receptors than previously thought, and neurons within the CNS are thought to express CB2Rs early in development before making a switch to solely expressing CB1Rs78. CB2R expression is also expressed in neuronal progenitor cells within the subgranular zone of the dentate gyrus of the hippocampus, and is thought to promote progenitor proliferation, showing impairment in CB2R-deficient mice79. CB2R expression is also found within embryonic stem cells80,81.

There is evidence the endocannabinoid anandamide also binds to and activates the transient receptor potential cation subfamily V member 1 (TRPV1) receptor82, and the endocannabinoid N-arachidonoyl-dopamine (NADA) has also been found to be a TRPV1 agonist83.

The orphan receptor G protein-coupled receptor 55 (GPR55) exhibits low homology (10-15%) to cloned CB1 and CB2Rs84. GPR55 has less than 20% sequence homology to either CB1R or CB2R, and the potency and efficacy of cannabinoids to GPR55 are highly disputed85,86. The peroxisome proliferator-activated receptors (PPARs) may also interact with endocannabinoids, but this has yet to be fully characterized as well87. These receptor types either have primary ligands other than endocannabinoids, or their binding of cannabinoids are not well characterized. Due to these reasons, we informally refer to these receptors as cannabinoid targets rather than cannabinoid receptors.

The activation of cannabinoid receptors may also cause direct or indirect activation of a number of other receptor types. Heteromeric complexes between CB1Rs and other GPCRs have been found, where a CB1R agonist was shown to reduce the affinity of a D2 receptor agonist binding within the dorsal and ventral striatum88. CB1R signaling was also found to be dependent on adenosine A2A receptor activation within the striatum, and blocking A2A receptor activation prevented the motor depressant effects of striatal CB1R activation89. CB1Rs may also form heteromultimers with orexin OX1Rs in cell culture90. The coupling of CB1Rs to other receptor types exemplifies the diverse roles cannabinoids play in the mammalian nervous system.

1.5.2 Cannabinoid receptor CNS distribution

There are high levels of CB1R binding and CB1R mRNA levels throughout the adult rat brain, especially within the striatum, cerebral cortex, hippocampus, septum, cerebellum, and brainstem91,92. The widespread expression of CB1Rs within the adult brain suggests endocannabinoids have an important role in normal brain function. In contrast, CB2Rs have been traditionally considered to be confined to the peripheral nervous system. However, it is now widely understood that CB2Rs are quite prominently expressed within microglia and are thought to play a role in CNS immune function93. For example, CB2R agonists inhibit dopamine neuronal firing within the ventral tegmental area, an effect absent in CB2R-/- mice94. Additionally, CB2R mRNA has been detected within the cerebellum95 and cultured granule cells96. Unfortunately, there currently appears to be few specific antibodies for CB2Rs, which often makes interpretation of immunoreactive labeling questionable97. Yet the discovery prominent CB1Rs and CB2R expression, found by a combination of immunohistological and electrophysiological techniques, suggests the endocannabinoid system plays a widespread role in synaptic function.

In addition to the canonical cannabinoid receptors CB1 and CB2, the cerebellum appears to express TRPV1 receptors. In situ hybridization in the cerebellum shows faint expression, although previous work has shown cerebellar western blot immunoreactivity98 and ELIZA expression99 for TRPV1. Despite discovering TRPV1 mRNA and protein expression within the cerebellum, there are no reported functional TRPV1 studies, which may suggest a lack of synaptic

TRPV1 modulation within the cerebellar cortex. If this were accurate it would suggest

endocannabinoids such as anandamide may exclusively activate classical cannabinoid receptors within the cerebellar cortex.

There is also evidence that GPR55 is expressed within the cerebellum and plays a role in motor coordination100, and our lab’s own preliminary data may support this. While whole-cell patch clamp recording from mouse granule cells, the GPR55 antagonist cannabidiol (CBD) increases sIPSC frequency onto granule cells, suggesting upstream GPR55 activation on Golgi cells. This is a plausible explanation since GPR55 is coupled with G-proteins Gαq/12 and Gα13, which increase intracellular [Ca2+] and leads to ERK phosphorylation101,102. Further studies are required in order to definitively confirm GPR55 expression, but the possibility of GPR55 expression in the cerebellar cortex provides an exciting way in which endocannabinoids could mediate cerebellar function.

1.5.3 Mechanisms of cannabinoid receptors

Cannabinoid receptors are part of the seven-transmembrane domain G protein-coupled receptor

(GPCR) family and are primarily coupled to Gαi/o heterotrimeric G proteins. When activated, the

Gi/o protein alpha subunit inhibits adenylyl cyclase and stimulates mitogen-activated protein kinases (MAPK), leading to decreased cAMP, PKA, and ultimately preventing calcium influx into the cell103. The βγ G-protein subunit is also known to lead to the inhibition of P/Q, N, and L-type voltage-gated calcium channels (VGCCs) and the stimulation of inwardly rectifying potassium channels (GIRK), leading to potassium efflux and hyperpolarizing the presynaptic terminal104–107.

Since vesicle release is dependent on calcium to bind SNARE proteins to fuse with the cell

membrane, activating CB1Rs effectively reduces vesicle release into the synapse. However, there is also some evidence suggesting CB1R pairing to Gs excitatory proteins, with an increase in cAMP

103,108,109 accumulation following CB1R activation . Even if Gs coupled CB1Rs are sparse, the ability for CB1Rs to diversify their function highlights endocannabinoid system adaptability.

CB1R activation can also activate the mechanistic target of rapamycin (mTOR) pathway110,111, which is capable of controlling protein synthesis and synaptic plasticity. CB1R activation of the mTOR pathway in the hippocampus is associated with hippocampal amnesic properties of THC, and blocking mTOR counters this effect112. Cannabinoids have also been linked to the activation of both ERK and FAK kinases113,114, and intracellular interactions with β-arrestins leads to CB1R desensitization115. The nature of CB1R activation, and their association with G- proteins, provides additional mechanistic pathways including those mentioned to provide unique downstream effects. This may act as an adaptable method in which the brain can cater to regulating abnormal synaptic signaling according to synaptic connections and excitability.

Postsynaptic TRPV1 receptors within the mouse and rat dentate gyrus have been observed to be activated by anandamide independent of CB1Rs, and can directly lead to long- term depression via the internalization of AMPA receptors in a Ca2+-calcineurin and clatherin- dependent manner82. Anandamide has been shown to be produced presynaptically to activate

TRPV1Rs, and may be inactivated by postsynaptic fatty acid amide hydrolase (FAAH), as FAAH has been observed on Purkinje cell soma and dendrites116. There is also some evidence that CB1Rs expressed on astrocytes are coupled to Gq/11 G-proteins, and can activate phospholipase C and

2+ produce inositol triphosphate (IP3), which leads to mobilization of internal Ca stores and stimulates glutamate release117.

1.5.4 Cannabinoids

There are a variety of endogenous and exogenous molecules that bind to cannabinoid receptors in the mammalian central and peripheral nervous systems. Plant-derived exogenous cannabinoid compounds (e.g. the cannabinoid THC originally isolated from cannabis in 19647) are typically referred to as phytocannabinoids and are responsible for cannabis’ psychopharmacological effects, such as time distortion, euphoria, relaxation and intensification of sensory experiences118. The plant contains more than 100 known phytocannabinoids2, but has higher percentages of those such as THC, cannabidiol (CBD), cannabinol, cannabigerol, etc.119. Though not as famous as its cousin THC, CBD has also been studied for its potential therapeutic properties, and may be considered a prime therapeutic candidate due to its primary actions on

CB2Rs within the peripheral nervous system and its lack of psychoactive effects119. It also binds to the orphan receptor GPR55, which is thought to be responsible for antiepileptic properties of

CBD120. Many phytocannabinoids have low affinities to cannabinoid receptors, and are often non- psychoactive, but nonetheless may influence endocannabinoid function. For example, cannabigerol has low receptor affinity, but is able to inhibit reuptake of the endocannabinoid anandamide121.

In addition to plant-based cannabinoid compounds, there are a number of synthetically produced exogenous cannabinoids, such as the compound WIN 55,212-2. These are primarily used for research purposes, but have also been found within the cannabis street alternative

“spice”122. The first synthesized CB1R antagonist was SR141716 (rimonabant)123, which was

briefly marketed by Sanofi Pharmaceuticals as an antiobesity drug in Europe before being withdrawn from the market for its psychological side effects in 2008. All of these cannabinoids consist of long-chain polyunsaturated fatty acids, and are highly lipophilic.

The mammalian nervous system has been found to produce two main endocannabinoids:

2-arachidonoylglycerol (2-AG), the most abundant endocannabinoid within the mammalian nervous system124, and anandamide (AEA), both arachidonic acid metabolites. 2-AG is postsynaptically synthesized from diacylglyerol (DAG) via sn-1-diacylglycerol lipase-α (DAGLα) and then inactivated mainly by presynaptic monoacylglycerol lipase (MAGL). AEA can be produced either presynaptically or postsynaptically by N-arachidonoylethanolamine-selective phospholipase D (NAPE-PLD) from N-arachidonoly phopshatidylethanolamine (NAPE) and then degraded by postsynaptic fatty acid amide hydrolase (FAAH)125. AEA is considered a partial cannabinoid receptor agonist, with higher affinity for the CB1R and CB2Rs, while 2-AG is a full agonist of both receptor types, yet exhibits lower affinity126. Both endocannabinoids are commonly produced in response to a rapid increase in postsynaptic intracellular calcium, hence its role as a synaptic modulator.

It is believed that there are likely other endogenous lipids found within the CNS that bind to cannabinoid receptors. An example is hemopressin, which is thought to be an endogenous

CB1R antagonist/inverse agonist127. Other suspected endogenous CB1R agonists are noladin ether (2-arachidonyl glycerol ether)128, CB1R agonist NADA83, and suspected CB1R antagonist virodhamine (O-arachidonoyl ethanolamine)129. However, the existence and role of some of these putative endogenous ligands is still disputed. Additional endogenous compounds are

suspected to interact with CB1R/CB2Rs. These include palmitoylethanolamide (PEA), an agonist of both CB1R and CB2Rs, and oleylethanolamide (OEA), a low affinity CB1R agonist130.

The differences in affinity of cannabinoids to cannabinoid receptors varies greatly. A meta-analysis compared the binding affinity of various cannabinoids and found Ki values (in nM) for: THC (HsCB1 = 25.1; RnCB1 = 42.6; HsCB2 = 35.3; RnCB2 = 13.0); CBD (RnCB1 = 2210.5; HsCB2

= 2860; RnCB2 = 1000); Anandamide (HsCB1 = 239.2; RnCB1 = 87.7; HsCB2 = 439.5); 2-AG (HsCB1

= 3423.6; RnCB1 = 1180.5; HsCB2 = 1193.8; RnCB2 = 1900). The Kd values (in nM) were determined for: WIN 55,212-2 (HsCB1 = 16.7; RnCB1 = 2.4; HsCB2 = 3.7; RnCB2 = 35.6);

SR141716A (HsCB1 = 2.9; RnCB1 = 1.0)131. Modeling studies based on the CB1R crystal structure predict that WIN binds deeper within the binding pocket than THC75, which may partially explain the higher receptor affinity.

1.5.5 Role of endocannabinoid system in synaptic modulation

The primary role of endocannabinoids appears to reduce high levels of synaptic vesicle release following postsynaptic depolarization, and has been found to mediate activity-dependent synaptic plasticity that may form transient and long-lasting synaptic depression in numerous types of synapses132. For example, cannabinoids have been reported to transiently suppress transmitter release through depolarization-induced suppression of inhibition (DSI; suppression of inhibitory vesicle release) and excitation (DSE; suppression of excitatory vesicle release), or to persistently suppress transmitter release through long-term depression86. Endocannabinoid- induced DSI was originally reported before DSE within stellate/basket cell synapses onto Purkinje

cells, where a depolarizing voltage train in Purkinje cells would reduce both the amplitude of spontaneous133 and evoked134 synaptic currents135 (see Diana & Marty, 2004 for good DSI/DSE review). Depolarization-induced endocannabinoid production was also found to explain the reduced excitatory parallel fiber and climbing fiber spontaneous activity in DSE experiments136,137. These phenomena are caused by increased levels of intracellular calcium in the postsynaptic terminal, where enzymes DAGLα and NAPE-PLD produce 2-AG and anandamide, respectively126. Although there appears to be transporters that shuttle endocannabinoids intracellularly138, there is a gap in knowledge regarding how endocannabinoids are shuttled from the postsynaptic terminal across the membrane to the extracellular space139,140. Depolarization- induced production of postsynaptic endocannabinoids leads to retrograde activation of presynaptic CB1Rs, which ultimately reduces vesicular release into the synapse. These actions provide additional fine tuning of synaptic transmission through means of presynaptic plasticity— neurons have a means of reducing excessive synaptic signaling following their depolarization.

Interestingly, cannabinoids have also been found to occasionally act as autocrine signaling molecules in addition to paracrine signaling87. For example, the endocannabinoid system can act as an autocrine signaling modulator within the adult cerebral cortex: a particular type of neocortical interneuron undergoes self-inhibition through increased K+ channel conductance, which is mediated by increased intracellular [Ca2+] and is blocked by the CB1R agonist AM251141.

This is in addition to its autocrine role within the developing brain (discussed in the following section, 1.5.7), and further exemplifies a unique endocannabinoid method of modulating neuronal excitability.

1.5.6 Role of endocannabinoid system in neurodevelopment

The endogenous cannabinoid system plays an important role in the developing nervous system.

CB1Rs and CB2Rs have been found on developing neurons, and cannabinoid exposure can alter neuronal development through their activation87. Previous studies have found functional CB1Rs in fetal rat brains as early as gestational day 14 (GD14), and CB1R mRNA has been found within discrete brain regions such as the hippocampus, cerebellum, and caudate-putamen at GD16142.

In human fetal brains, CB1R expression has been reported as early as the 14th gestational week, with high expression in the cerebral cortex, hippocampus, caudate nucleus, putamen, and cerebellar cortex; functional CB1Rs are found within human brains at 19 weeks of gestation within these same brain regions and the frontal cortex143.

Endocannabinoid signaling within the telencephalon begins as soon as embryonic day 12 in mice, and control proliferation of pyramidal cell progenitors and radial migration of immature pyramidal cells144. The simultaneous expression of CB2R and DAGLα/DAGLβ—the enzymes responsible for 2-AG synthesis—in neural progenitor cells suggest autocrine endocannabinoid signaling may support asymmetrical cell division, cell cycle exit, and long-range migration of ensuing progenies78. CB1Rs are also believed to increase neuronal precursor proliferation through AKT/Glycogen synthase kinase-3β/β-Catenin signaling in cerebellar granule cells145, and

CB1R expression is found in premigratory GABAergic interneurons within the hippocampus, which may promote neuronal polarization and commencement of long-range cell migration146.

In addition, CB1Rs are enriched in the axonal growth cones of GABAergic cortical interneurons during late gestation, and endocannabinoids can lead to filopodia CB1R internalization and to

axonal growth cone repulsion and collapse147, a mechanism which has also been observed through contraction of the actomyosin cytoskeleton by Rho-GTPase and Rho-associated kinase

148 (ROCK) through G12/13 G-proteins . There is also evidence that endocannabinoids can be used to alter cortical oscillations in the developing cerebral cortex by controlling the firing of inhibitory interneurons via an autocrine mechanism141, which may influence cerebellar network activities in a similar way.

CB1R-mediated neurodevelopment may manifest through unique signaling pathways. In

CB1R-transfected embryonic kidney cells, CB1R activation induces cell migration, which is inhibited with the use of a MAP kinase inhibitor, but not a cAMP analog, suggesting the G protein- induced adenylate cyclase cascade is not as important for migration, in this case149. The role of endocannabinoids as migratory signaling molecules is further exemplified by high levels of MAGL

(2-AG degradation enzyme) localization used to limit axonal spread in the “prospective” internal capsule and their “delineation” of migratory routes for CB1R-expressing cortical interneurons150.

This suggests that endocannabinoid levels must be highly localized and controlled to ensure proper migratory patterns, and promotes the concept of endocannabinoid “hot spots” that exist for focal signaling events to drive migration and axonal elongation in neurons146. Physiologically relevant cannabinoid concentrations could affect large numbers of migrating neurons or axons during pathfinding since the spatial spread of endocannabinoids in the fetal brain is less restricted than in the adult brain151. Thus, disrupting both the localization and relative concentration of cannabinoid signaling could have developmental consequences.

Also, localization of cannabinoid receptors may determine how they are able to influence neurodevelopment. For example, cannabinoid receptors are not simply isolated to neurons, as

CB2R activation on microglia and CB1R activation on astrocytes have been found to regulate various cell functions, such as increase microglial cell proliferation and migration152, or stimulate astrocyte glutamate release117. Blocking glutamate uptake, potentially from astrocytes, increases spontaneous NMDA channel openings in cerebellar granule cells45 and increases granule cell migration rate in the molecular layer40, which potentially implicates glial cell cannabinoid receptor activation in modulating neurodevelopment.

The endocannabinoid system may also indirectly modulate developmental signaling molecules, and the types and/or combinations of guidance cues may vary by cell type. For example, there may be an interaction between cannabinoid signaling and glutamatergic signaling in the migration of immature cerebellar granule cells, as glutamate has been shown to be an integral migratory cell-signaling molecule45,153, and disproportionately high CB1R activity is able to dampen NMDAR activity10, which may support an association between CB1R activation and glutamatergic guidance cue disruption.

Transitory developmental expression of endocannabinoids, eCBRs, and eCB enzymes.

The transitory expression and positional differences of endocannabinoid enzyme expression within the CNS during brain development has been well characterized. DAGLα, the enzyme responsible for synthesizing 2-AG, is coexpressed with CB1Rs axonally in pyramidal cell progenitor pathfinding growth cones before transitioning to a postsynaptic locale following neuronal differentiation144. The 2-AG-degrading enzyme monoacylglycerol lipase (MAGL) is also coexpressed with DAGLα and CB1Rs on corticofugal pyramidal cell axons, but absent from the neurite tip, suggesting endocannabinoids perform an autocrine pathfinding role in promoting

axonal growth154. Both DAGLα and DAGLβ are shown to be expressed in spinal axons on embryonic day 10 (E10) in mice and DAGLβ is seen in PND 14 retinal ganglion fiber tract and optic nerve, but expression of both enzymes are absent in adult mouse tissue155. In adult cerebellar cortex, the highest levels of DAGLα and DAGLβ expression are seen in Purkinje dendritic trees, but are absent from axonal tracts, where DAGLβ is severely downregulated in adulthood compared to DAGLα155. These results suggest the transitory expression of proteins associated with the endocannabinoid system, which provides evidence showing the endocannabinoid system is highly adaptive to accommodate the mechanistic needs of the developing brain beyond the scope of synaptic modulation.

There is also evidence to suggest neurons transition from primarily expressing CB2Rs before switching to CB1Rs following cell differentiation. Researchers reveal a CB2R agonist, but not a CB1R agonist, increases neurogenesis in the subventricular zone (SVZ) in 6-month-old mice; using WIN 55,212-2, a CB1R and CB2R agonist, neurogenesis was again increased, but this effect was completely blocked by the addition of a CB2R antagonist78. Interestingly, these manipulations in 20-month-old mice led to neurogenesis levels equivalent to 6-month-old mice, showing a clear effect of age on cannabinoid-induced neurogenesis. This mechanism may expand beyond the SVZ, and be found in additional brain regions such as the cerebellum.

1.6 Endocannabinoid System within the Cerebellum

1.6.1 Cannabinoids in adult cerebellar cortex

The role of endocannabinoids in the modulation of Purkinje cell activity is fairly well characterized. Depolarization-induced suppression of inhibition and excitation (DSI & DSE) is a common phenomenon seen following Purkinje cell depolarization. Endocannabinoid production responsible for DSI and DSE are produced in Purkinje cell postsynaptic sites and bind with presynaptic CB1Rs on parallel fibers and stellate/basket axons, which reduces their respective vesicle release probability133,134,136,137. This knowledge has been developed using electrophysiological techniques, but other studies have attempted to elucidate CB1R locations using other methods. Many studies have used immunohistological techniques to elucidate cannabinoid receptor expression. Cannabinoid receptors within the cerebellum have been generally considered to be limited to the molecular layer and on basket cell terminals within the cerebellar cortex, while Purkinje cells themselves seem to lack CB1R expression156–158.

Furthermore, there has been little to suggest expression on granule cells, Golgi cells, or mossy fibers, as they seem to be devoid of immunoreactivity. There have been a number of studies using in situ hybridization in order to identify CB1R gene expression within the cerebellar cortex.

Granule cells display high levels of CB1R mRNA expression159, although this is generally interpreted to be localized to granule cell parallel fibers within the molecular layer when combined with immunohistochemical data. However, one group reported what they thought to be expression among a sparse number of granule cells160. Upon closer examination of the data, the reported CB1R immunoreactivity may be localized to the glomeruli that make up granule cell

synapses between Golgi cells and mossy fibers, or on Golgi cells themselves. CB1R expression does not appear to overlap with GLAST staining in PND 21-28 rats, which is a marker of Bergmann glial fibers161, which would suggest no glial expression of CB1Rs at this age.

In addition to the aforementioned studies regarding DSI and DSE, a number of studies have investigated adult CB1R activity in the cerebellum using electrophysiological techniques.

The addition of the CB1R agonist WIN 55,212-2 (WIN) has reduced Purkinje cell parallel fiber eEPSC amplitude, reduced parallel fiber-evoked asynchronous events, decreased mEPSC frequency, and impairs LTD by decreasing the probability of glutamate release162,163. Suppression of parallel fiber EPSCs by mGluR1 agonists was only partly blocked by CB1R antagonists in PND

18-22, compared to a full block in PND 10-12 rats164. They concluded the mature synapses also contain presynaptic kainite receptors located on PFs and displayed prolonged release of calcium from presynaptic internal calcium stores when activated by glutamate. WIN also reduces inhibitory GABAergic interneuron163 and climbing fiber136,137,161,163 signaling onto Purkinje cells.

Some of the CB1Rs on GABAergic inhibitory terminals may be constituently active as well, revealing a tonic CB1R-driven inhibition of vesicle release. For example, CB1R antagonist AM251 and inverse agonist SR141716A increase mIPSC frequency in PND 9-14 mouse165 and PND 14-21 rat166, although this was not replicated in a different study using PND 18 rats167.

Cerebellar-associated behavioral effects

It has been long understood that cannabis effects fine motor skills in humans, and a number of behavioral studies have also confirmed the importance of cannabinoid signaling within the cerebellum. Human motor control performance studies revealed consumption of high-

potency cannabis (13% THC) impaired performance in a Critical Tracking Task168. THC impairs motor coordination, decreasing motor activity in mice and producing ataxia in dogs169. Rats exposed to CB1R agonist WIN display delayed motor learning; climbing fiber firing has been known to have a role in encoding eyeblink and motor learning, so the delayed motor learning may be caused by climbing fiber misfiring170. Intracerebellar microinjections of CB1R agonists

CP55,940 and HU-210 created dose-dependent motor incoordination in 5-6 week old mice171.

Also, CB1R agonist injections produced behavioral deficiencies associated with disrupted cerebellar function in mice, such as increased gait width (truncal ataxia) and slips on the bar cross test. These results may be cerebellar specific because pretreatment with dopamine receptor agonists failed to rescue the behavioral deficiencies, which would suggest cannabinoid- alterations did not occur in the basal ganglia or be caused by alterations in nigrostriatal dopamine transmission172. These behavioral data, when combined with immunohistochemical and electrophysiological data, strongly suggest cannabinoid receptors within the cerebellar cortex are able to modulate cerebellar-associated behavioral tasks.

1.6.2 Cannabinoids in developing cerebellar cortex

Studies examining the cannabinoid system within the developing cerebellar cortex have been sparse, with the majority being carried out in cell autonomous preparations, such as in cerebellar granule cell cultures. Among these studies, high immunohistochemical CB1R reactivity has been found on granule cell plasma membranes, including granule cell neurites between cell clusters173.

Cultured cerebellar granule cells have also responded to CB1R agonists THC174,175 and WIN

55,212-2173 in PND 6-8 rat pups. CB1R activation in granule cells have been observed to trigger intracellular signaling cascades (inhibition of Ca2+ influx inhibition, nNOS activation, and cAMP/Epac2/PLC pathway) in primary cultures of developing rat cerebellum (PND 6-8, PND 6-8,

& PND 7, respectively)173,176,177. In addition, cell cultures from PND 6-8 rat pups exhibited reduced intracellular Ca2+ influx through voltage-gated calcium channels following application of CB1R agonists CP55940, WIN 55,212-2, and anandamide, which was abolished in the presence of the

G protein blocker pertussis toxin173. These data are insightful in revealing CB1R expression on developing granule cells, but unfortunately provide little information regarding the functional role of CB1Rs in synaptic signaling, and how CB1R activation can ultimately influence developing cerebellar synaptic transmission.

Although most developmental cerebellar experiments have been conducted in cultured granule cells, a number of cerebellar cannabinoid papers include experiments on developing

Purkinje cells in young animals due to the difficulty in patch-recording from large, older Purkinje cells. For example, Purkinje cell mGluR1 receptor activation in PND 8-13 mouse Purkinje cells appear to produce endocannabinoids since mGluR1-activation lead to a reduction in climbing fiber neurotransmitter release, an effect blocked by CB1R antagonists178. Others have found that administration of mGluR1 receptor agonists reduces parallel fiber evoked EPSCs in PND 10-12 rat

Purkinje cells, an effect that is blocked using a CB1R antagonist164. However, in a separate study,

CB1R expression in mice does not appear to colocalize with VGluT1 (parallel fiber terminals) at

PND 14, and VGluT2, known to be expressed in both climbing fiber and immature parallel fiber terminals in developing cerebellum, did not overlap with CB1Rs at higher magnification178. This would suggest a lack of CB1R expression at glutamatergic synaptic terminals, in contrast to

electrophysiological experiments. Also, in addition to glutamatergic synapses, there was overlap with the vesicular GABA transporter VGAT along inhibitory fibers and terminals around Purkinje cell somata178, suggesting CB1R expression at GABAergic terminals. These experiments suggest that CB1Rs may begin to be expressed on Purkinje afferents towards the end of our developmental window, and may perhaps be functionally expressed at an earlier time period.

1.7 Effects of Neurodevelopmental Cannabinoid Exposure

There have been a number of human longitudinal and rodent studies examining the effects of chronic exogenous cannabinoid exposure on neurodevelopment and its resulting consequences on behavior. Because of the lipophilic nature of the phytocannabinoid THC, the most abundant cannabinoid found within cannabis, it is estimated that one-third of THC in the plasma crosses the fetoplacental barrier179 and is secreted in the breast milk180. Exogenous cannabinoids also reside within mammalian tissue for an extended period of time, where THC and its metabolite

11-hydroxy-THC are found in breastmilk up to roughly six days after last reported use181. These results make clear that fetuses and babies can be indirectly exposed to THC, but do not examine whether THC is able to access the brain. However, THC is found in the fetal plasma and brain of rhesus monkeys, suggesting it crosses the blood brain barrier182. This method of THC exposure may produce neurodevelopmental effects. For example, cannabis exposure in mid-gestational human fetuses leads to a reduction in foot length and body weight; fetal foot length negatively correlated with the amount and frequency of cannabis use reported by their mothers183. Also, maternal cannabis use increased rates of neonatal morbidity such as pulmonary morbidity and

infection184. These findings suggest that multiple organ systems may be vulnerable to exogenous cannabinoid exposure during early development.

The majority of human studies have focused on the effects of exogenous cannabinoids on complex cognitive tasks involving attention, learning and memory, emotional behavior, and some motor function. We should note that researchers are beginning to appreciate the role the cerebellum may play in developing a number of neurodevelopmental disorders15,17,18,185,186, so cerebellar dysfunction may be contributing to the cognitive and emotional reward processing deficiencies seen as well. While most studies have focused on these abilities from a behavioral level, the number of studies examining the cellular effects of cannabinoids and how alterations in their function may explain such behavioral changes have been sparse. The remainder of this section focuses on some of the brain regions thought to be most impacted by exogenous cannabinoid exposure during early neurodevelopment and the neurobehavioral consequences that result.

1.7.1 Motor Function

Alternations in motor function constitutes one of the main behavioral effects following cannabis consumption. As such, researchers have been interested in the effects of exogenous cannabinoids on cerebellar function. Most clear correlations between cannabis exposure and behavioral deficiencies have been short-term, so long-term changes in motor function due to neurodevelopmental cannabis exposure has been somewhat controversial. A number of studies,

including those following, are in disagreement as to whether there are any motor issues in offspring that result from pregnant mother cannabis consumption.

There have been reports that cannabinoid exposure during pregnancy and/or lactation in rats leads to motor hyperactivity during infancy and adolescence, but these effects fail to endure into adulthood187,188. In contrast, an early human study found that postpartum maternal cannabis use impaired 12 month old infant motor development through THC concentrated breast milk189.

Some of the effects of cannabis use may persist into adolescence, with defects in processing speed on a bimanual coordination task reported in the 16 year old children of women who consumed cannabis during pregnancy6. Animal studies have found that treating pregnant rats with an exogenous cannabinoid leads to motor function impairment in postnatal day 22 (PND 22) but not PND 36 offspring, while rearing frequency was reduced in PND 22, 36, and 50 offspring68.

Interestingly, exposing PND 4-9 rat pups to CB1R agonist CP-55,940 appeared to improve early motor development (hindlimb coordination; PND 12-20), but did not affect long term motor ability190.

From an anatomical level, prenatal WIN consumption in rats was associated with atrophy of the granule cell layer and Purkinje layer of the cerebellar cortex, while concurrently leading to a decrease in rearing frequency of the offspring and an increase in grooming frequency191.

However, these results failed to translate to a separate study, where pregnant rats exposed to an exogenous cannabinoid produced offspring (PND 120-150) with no structural alterations in the cerebellar cortex, and also displayed normal GAD/GABA distribution (representing normal inhibitory GABAergic synapses); yet, the magnitude of GAD/GABA expression was significantly increased in the molecular and Purkinje cell layers, but not in the granular layer192. It is difficult

to speculate why cannabinoid expression may increase supposed GABA expression and/or transmission, but altering GABAergic transmission could theoretically alter intrinsic Purkinje cell firing properties.

1.7.2 Non-motor Behavioral Effects

Executive function.

There has been a recent surge in studies examining the effects of cortical cannabinoid exposure during neurodevelopment. Prenatally exposing rats to the CB1R agonist WIN 55,212-2 alters glutamatergic and GABAergic neuronal migration in the cerebral cortex193 and leads to deficiencies in cortical NMDA signaling, coupled with performance issues in emotional reactivity tasks194. Adolescent rats (PND 30-60) exposed to the cannabinoid receptor agonist WIN 55,212-

2 underperform in mPFC-dependent Probabilistic Reward choice tasks195. Additionally, exposing adolescent rats to the cannabinoid (−)-trans-Δ9-tetrahydrocannabinol (THC) leads to the impairment of both cannabinoid signaling and long-term depression (LTD) in the prefrontal cortex196, while cannabis exposure in fetal sheep leads to a reduction in EEG delta power and delta distribution197. In humans, prenatal marijuana exposure impairs attentional behavior and visual analysis/hypothesis testing198, and a longitudinal study 10 years post-pregnancy found 10 year old children whose mothers consumed more than one cannabis joint a day while pregnant displayed deficits in reading and spelling scores. This was accompanied by lower teachers’ evaluation rating on the children’s performance, after taking into account differences in home environment and socioeconomic status199.

Learning and memory.

The short-term effects of exogenous cannabinoids on hippocampal functioning are well documented, and makes it a model system due to the high density of CB1Rs within the region and its associated psychoactive effects following cannabis consumption92,200. Short-term memory deficits are a well-known effect of cannabis consumption, suggesting a role for cannabinoid actions in the hippocampus201,202. Due to the pronounced effects of cannabis consumption on short-term hippocampal function, there may also be a strong endocannabinoid role in hippocampal neurodevelopment. In support of this claim, pregnant rats exposed to the CB1R agonist WIN55-212,2 gave birth to offspring that displayed disrupted memory retention when subjected to a passive avoidance task, which was coupled with alterations in hippocampal long- term potentiation and glutamate release within CA3-CA1 synapses187. Also, there is decreased functional activity and expression of glutamate transporters GLT1 and GLAST in the hippocampus of rats perinatally exposed to THC203. PND 4-9 rat pup exposure to CB1R agonist CP-55,940 produced disruptions in future behavioral tasks such as open field tests (PND 18-21), and causing altered thigmotaxis during the Morris water maze (PND 25)204. These data clearly support an endocannabinoid role in neurodevelopmental processes that translate to memory associated behavioral changes following exogenous cannabinoid exposure during development.

Although memory-related changes following exogenous cannabinoid exposure appear to be less controversial, one reason many of these studies may be in disagreement may stem from the various protocols used, such as time of exposure, route of administration, cannabinoid compound used, and age of testing205. Undoubtedly, the majority of the rodent studies examining the developmental consequences of exogenous cannabinoid exposure administer the drug

during gestation, a period not in parallel with human third trimester pregnancy (discussed in section 1.4.4: Rodent age equivalent of third trimester human fetuses), which may convolute our associations between animal and human studies comparing gestational and adolescent cannabinoid exposure.

1.8 Neurocognitive Disorders Associated with Cerebellar Dysfunction

There have been various cognitive disorders associated with cerebellar dysfunction, where the majority of these findings have been through associative conclusions drawn from humans born with cerebellar damage or from adults who have suffered specific cerebellar injury (e.g. stroke).

The most compelling aspect of these disorders arises from the non-motor symptomology, which suggests the cerebellum is involved in higher-order processing. Individuals with cerebellar lesions, primarily within the posterior lobe and vermis, exhibit a particular range of cognitive symptoms that have been defined as cerebellar cognitive affective syndrome31. Executive function impairments include deficits in planning, set-shifting, verbal fluency, abstract reasoning, and working memory; spatial cognitive deficits include difficulties with visual-spatial organization and memory; also present are personality changes with blunted affect, disinhibited and inappropriate behavior, and language deficits31,206.

The neurodevelopmental disorder autism spectrum disorder (ASD), as discussed in the subsequent section, likely arises from cerebellar dysfunction during brain development. It is possible altered cerebellar circuitry driven by drug exposure could produce similar behavioral deficiencies, and drug-induced alterations in cerebellar circuitry may include exposure to exogenous cannabinoids during neurodevelopment. While ASD is unlikely to be primarily caused

by drug exposure, many of these cognitive symptoms resemble those seen following neurodevelopmental exposure to exogenous cannabinoids, such as attentional behavior and visual analysis/hypothesis testing198 (see Section 1.7.2: Other Long-Term Behavioral Effects).

1.8.1 Autism Spectrum disorder

One of the most compelling arguments for a role of the cerebellum in cognitive development is the close correlation between cerebellar damage and the development of autism spectrum disorders (ASDs). Early disruption of the cerebellar circuitry has been shown to be positively correlated with autism207–210, and cerebellar damage at birth is the leading environmental predictor of developing autism at a later age15. Various labs have attempted to identify ASD- related gene disruption. Many of the coexpressed ASD susceptibility genes identified are involved in various stages of neuronal development, including neuronal differentiation211. What is interesting is that these genes coexpress during two distinct periods of development known to be critical periods in which brain trauma can supposedly lead to autism-like symptoms. In addition, many of these coexpressed susceptibility genes are involved in synaptic plasticity, development, and neuronal differentiation211, suggesting that these genes may disrupt cerebellar circuitry and aid in the development of autism-like symptoms15. It is believed damaging the cerebro-cerebellar circuit, a network of closed-loop circuits that may be involved in communicating with cognitive and emotional affective cortical regions, may have an impact on the development of ASD212. This would coincide with the theory of ASD as a disorder that manifests through the inability to make cognitive predictions of events, an ability that is

intimately reliant on proper cerebellar function213. Cerebellar dysfunction has also been linked to sensory learning deficits found in mouse ASD models15,214. Future research will further refine our understanding of the role the cerebellum may hold in the manifestation of ASD-like symptoms, and how developmental perturbation on cerebellar anatomy and synaptic signaling may be partially responsible.

1.9 Therapeutic Effects of Exogenous Cannabinoids

Humans have been consuming cannabis for medicinal purposes based on anecdotal evidence long before understanding the molecular underpinnings guiding its effects. The Greek historian

Diodorus Siculus (60 B.C.) mentioned Egyptians used cannabis to reduce pain and improve mood, while the Roman Pliny the Elder (70 A.D.) used the roots for pain management2. Cannabinoids are still used to assuage pain and inflammation215,216, but controlled studies examining the therapeutic effects on pain management are often mixed217. The phytocannabinoid cannabidiol has been shown to host a variety of therapeutic effects on a number of disease states, including glaucoma and epilepsy. For example, the CB1R agonist WIN 55,212-2 reduces glaucoma intraocular pressure216,218, and cannabidiol reduces seizure phenotypes in a genetic mouse model of Dravet syndrome (DS), a childhood epilepsy disorder120. Cannabinoids have also shown promise in treating multiple sclerosis216,219,220, and CB1R antagonists have been considered for the treatment for obesity-related metabolic disorders221 and various forms of mental illness196,222.

Cannabinoids may serve to benefit several cellular ailments as well. Activating the endocannabinoid system has neuroprotective effects in cerebellar granule cells96, where

cannabinoids can counter remote cell death in the cerebellum following brain injury223. CB2R agonists specifically seem to display neuroprotective properties, having anti-apoptotic effects:

CB2Rs are upregulated following cerebellar hemispheric lesioning, and CB2R agonists reduced neuronal loss through a PI3K-dependent pathway223. These findings convey clear potential for cannabinoid therapeutics in the future, which may include neuroprotective uses in addition to those aforementioned.

1.10 Summary/Discussion

Despite recent advances in the understanding of cerebellar development and the role of the endocannabinoid system, there is still a great deal to be learned, understood, and refined. The scientific community has just recently begun to explore which proteins and molecules comprise the endocannabinoid system, and have begun to examine its range of effects on various neuronal systems. There is a wealth of evidence implicating the endocannabinoid system in a host of neurodevelopmental processes, although the majority of these findings have either occurred outside of the cerebellum or in dissociated neurons. This introduction has so far highlighted these findings, but has also conveyed that very little is known regarding the endocannabinoid systems influence over cerebellar development, and how perturbation may affect cerebellar processing.

The cerebellum undergoes the majority of its development during the third trimester of pregnancy to about 9 months post-utero in humans, and between about postnatal day 2-20 in rodents. Recent discoveries have implicated the endocannabinoid system as an important signaling modulatory system during neurodevelopment; CB1R activation has been shown to be implicated in cell proliferation, migration, and synaptogenesis in developing tissue. The

involvement of the endocannabinoid system in neurodevelopmental processing may implicate inappropriate cannabinoid signaling in abnormal structural and behavioral phenotypes. This makes the endocannabinoid system within the cerebellum an ideal study candidate in terms of how cannabinoids interact with receptors in a way that could impact developmental processes.

These reasons include having a firm grasp of normal cerebellar circuit functionality, and there are typically clear behavioral phenotypes associated with its perturbation. Yet we currently lack an understanding in regard to where cannabinoid receptors are expressed, if they are expressed at all, within the developing cerebellum.

Cerebellar function is clearly implicated in the coordination of classic motor function, including refinement and sensorimotor processing29. It also appears to have more nuanced roles in cognition, emotional affect, executive functions, and may be involved in the development of neurodevelopmental disorders due to the association between cerebellar damage and the development of abnormal phenotypes15,31. These associations may indicate exogenous cannabinoid exposure is detrimental to development during critical periods of cerebellar growth.

Phytocannabinoids found in cannabis such as THC and CBD are the most well-known exogenous effectors of the mammalian endocannabinoid system. Despite humans’ steadily consuming cannabis throughout recorded history, cannabis became predominantly illegal over the last 100 years. However, there has been a recent push towards its re-legalization as the popularity of cannabis for recreational and medicinal purposes has steadily grown. Expanding our knowledge into how the endocannabinoid system works, and the resulting impact of cannabis on its exerting effects on cannabinoid receptor activation, is especially relevant as legalization outpaces our understanding of potential health impacts. It is crucial to relate our

findings within this dissertation to the eventual development of potential guidelines in regards to maternal consumption of cannabis during pregnancy.

We are able to build on previous work within the cerebellum to develop a more complete understanding of the endocannabinoid system as it pertains to cell signaling. For example, CB1Rs are reportedly expressed on cultured granule cells, and CB1R activation hosts neurodevelopmental actions, which also guides our research interests. Yet taken together, there are few experiments collating these ideas to understand endocannabinoid function within the developing cerebellum. The experiments comprised within this dissertation attempt to characterize CB1R expression within the developing cerebellar cortex, the role CB1R activation has on synaptic signaling, and the ramifications of exogenous activation on cerebellar development.

CHAPTER 2

CB1R distribution in the cerebellar cortex throughout cerebellar development, and their role

in synaptic function.

Jesse L. Barnes and David J. Rossi

ABSTRACT

CB1Rs are one of the most abundant GPCRs in the mammalian nervous system, and are integral to modulating synaptic signaling ubiquitously throughout the brain. Within the mature cerebellar cortex, CB1Rs are found on both glutamatergic and GABAergic terminals presynaptic to Purkinje cells, which can act to influence output out of the cerebellar cortex. However, little is known regarding functional CB1R expression in developing (PND 4-12) cerebellar tissue in slice during a developmental period when the cerebellum in undergoing massive developmental changes. In this Chapter, we characterize abundant CB1R expression throughout all layers of the developing rodent cerebellar cortex using immunohistochemical labeling, and we systematically characterize functional CB1R activity electrophysiologically in all major neuronal types. We find that the CB1R agonist WIN 55,212-2 (WIN) reduces vesicle release probability in both glutamatergic and

GABAergic synaptic inputs onto granule cells by activating presynaptic CB1Rs, but WIN interestingly fails to reduce vesicle release probability onto developing Purkinje cells. This is opposed to previous findings in mature tissue where CB1R activation strongly attenuates

neurotransmitter release onto Purkinje cells, revealing a form of transient developmental CB1R expression. Additionally, we find GABAergic, but not glutamatergic, synaptic transmission responds to WIN in mature (PND 19-36) granule cells, suggesting mossy fibers express transiently restricted CB1R expression. Therefore, CB1R expression within the developing cerebellar cortex is developmentally unique. These data reveal that CB1R exhibits developmentally unique expression in multiple synapses within the cerebellar cortex and plays an active role in synaptic modulation during the most developmentally active period of cerebellar cortical growth.

INTRODUCTION

CB1Rs are the most abundant G-protein coupled receptor (GPCR) in the central nervous system, and comprise the majority of GPCRs in the brain92. A large proportion of CB1Rs are found within the cerebellar cortex126,142,156,200, and endocannabinoid signaling has been found to mediate a form of activity-dependent synaptic plasticity that may form transient and long-lasting synaptic depression in numerous types of synapses132. Within the mature cerebellum, the location of

CB1Rs presynaptic to Purkinje cells is fairly well characterized; CB1Rs have been found on excitatory glutamatergic granule cell parallel fibers and climbing fibers, and inhibitory interneurons Stellate and basket cells. In situ hybridization studies have shown heavy CB1R mRNA reactivity within the cerebellar cortex, which is especially dense within the granule cell layer224.

Immunohistochemical studies show heavy CB1R immunoreactivity within the molecular layer and what appear to be basket cells157. In situ hybridization studies reveal high CB1R mRNA levels within stellate, basket, and granule cells142,159. The high CB1R mRNA levels in granule cells may correspond to the heavy molecular layer CB1R protein expression seen in immunohistochemical

studies, which is presumably expressed on parallel fiber axons. When combined, these data suggest there is CB1R expression on Purkinje cell presynaptic terminals. To support this, electrophysiological studies show the CB1R agonist WIN 55,212-2 greatly reduces glutamatergic and GABAergic signaling onto Purkinje cells163. When depolarizing Purkinje cells, researchers have been able to induce endocannabinoid production and reduce vesicle release

(depolarization-induced suppression of excitation/inhibition)133,134,225 within these synapses.

Also, depolarizing Purkinje cells lead to reduced molecular layer interneuron firing through a K+ current, suggesting endocannabinoids can reduce synaptic vesicle release and reduce firing frequency of molecular layer interneurons166. Taken together, these studies indicate functional

CB1R expression presynaptic to Purkinje cells is able to reduce vesicle release into the synapse.

Yet despite the range of studies within these aforementioned synapses, little is known regarding whether CB1Rs are found upstream of Purkinje cells, how neuronal development influences their expression, and whether they function similarly as they do in mature Purkinje cell synapses.

Despite the wide range of cannabinoid experiments conducted in mature cerebellar tissue, there remains much to be learned regarding cerebellar cannabinoids during development.

Many, if not all, of the cerebellar developmental studies have been carried out in primary granule cell cultures from developing rodents. For example, a number of studies have found that cultured cerebellar granule cells from PND 6-8 rat pups exhibit either 1) blunted adenylyl cyclase activity or 2) reduced Ca2+ influx through voltage-gated calcium channels following administration of

CB1R agonists Δ9-tetrahydrocannabinol (THC)174,175, WIN 55,212-2, CP55940, and anandamide173.

These effects were abolished in the presence of pertussis toxin, revealing these effects were by activation of a G protein coupled receptor CB1. Also, high immunohistochemical CB1R reactivity

has been found throughout granule cell neuronal membranes, including on neurites173. In addition to the lack of knowledge pertaining to developmental cerebellar CB1R expression, to our knowledge no research has been done to determine CB1R expression in mature cerebellar granule cells. These gaps in knowledge are clear points of research needed to better characterize endocannabinoid functionality within the cerebellum as a whole.

We spent this chapter attempting to both visually localize CB1Rs in reference to other known proteins expressed in the cerebellum, and measure the role CB1Rs play in mediating synaptic activity. This was achieved during two distinct cerebellar developmental time points: 1) a developmental period (PND 2-12), which is a third trimester equivalent during the greatest period of cerebellar growth, and 2) a mature (PND 30-35) developmental period, in which all major cerebellar developmental processes have been achieved, such as granule cell migration and synaptogenesis. We outline immunohistochemical and electrophysiological techniques used in rodents to pinpoint CB1R location within the primary circuitry of the cerebellum. This will aid to better understand which cells express CB1Rs, reveal their role in synaptic transmission, and allow us to speculate on a developmental role of CB1Rs within the cerebellar cortex.

MATERIALS AND METHODS

Animals. All animals were bred and housed within a Washington State University (WSU) vivarium.

All procedures conformed to regulations approved by the Washington State University

Institutional Animal Care and Use Committee (IACUC), and conformed to all guidelines for ethical protocols and care of experimental animals established by the National Institutes of Health,

Maryland, USA. Either Sprague Dawley rats (Simonsen) or C57BL/6J transgenic CB1R knockout

mice (bred within the WSU vivarium; originally from Rockefeller University) were used for all experiments. The C57BL/J6 CB1R knockout mice were originally designed as described in Zimmer

& Bonner, 1999, in which the CB1 gene was mutated by replacing its coding sequence, which is confined to a single exon. The exon coding region of CB1 was replaced with PGK-neor through homologous recombination in embryonic stem cells, which completely blocks expression of the

CB1 exon. All CB1 knockout mice used for experiments were bred from heterozygous CB1+/- parents and were either homozygous CB1+/+ (for controls) or CB1-/-.

Histology tissue sectioning. Rodent cerebelli were extracted under 1°C aCSF bubbled with 95%

O2/5% CO2 gas and immediately placed into 4% paraformaldehyde solution and allowed to fix for

24 hours. Tissue subsequently underwent an antifreeze preparation in which they were placed in 20% sucrose solution until sinking, followed by 30% sucrose solution (typically a 24 hour process per solution). Cerebellar vermis tissue was then sectioned either in the sagittal or coronal plane at 40 µm using a Leica CM1950 cryostat with a -18°C chamber temperature. Sections were placed in a cryoprotectant solution (containing 0.1M sodium azide) and stored in a -20°C freezer until staining.

Immunohistochemistry. Cerebellar vermal sections were taken from cryoprotectant solution and underwent a series of free-floating 1X phosphate-buffered saline (PBS) washes before a 45 min blocking step in 5% bovine serum albumin (BSA) in 0.5% Triton X-100/PBS solution. Following a series of PBS washes, tissue was subjugated to a 20 min incubation containing nuclear stain

Hoechst 33342 (8.12 mM; Molecular Probes H1399) before another series of PBS washes and

finally into a 24 hour primary antibody incubation. The tissue was then washed and placed into a 2 hour secondary antibody incubation before a final series of PBS washes and mounted on microscope slides. The slides were then coverslipped using Permount Gold and allowed to dry until confocal imaging. All images are sagittal unless noted otherwise.

Confocal microscopy. Fluorescent images were acquired using a Leica SPX-8 white light point- scanning confocal microscope with Leica imaging software. Images were acquired using one of three objectives: HC PL APO 20X/0.7 NA, HC PL APO CS2 40X/1.3 NA (oil immersion), or an HC PL

APO CS2 63X/1.4 NA (oil immersion). Laser excitation wavelengths were taken at 488 (4% laser),

567 (5% laser), and 647 nm (2% laser); light was collected at 522-572 nm for Alexa Fluor 488, 602-

650 nm for Alexa Fluor 568, and 672-730 nm for Alexa Fluor 647. Hoechst 33342 nuclear stain was visualized using an ultraviolet bulb and light was collected at 450-500 nm. A separate sequence was used for Alexa Fluor 568, where only 568 laser and 602-650 nm emissions were collected in order to avoid inappropriate fluorescence collection, which was due to bleed- through from the ultraviolet bulb. Digital gain and photon emission light gating, the time set to detect fluorescence emission between light pulses, were identical for both species and conditions.

Acute preparation of brain slices. Rodent ages were either between postnatal day (PND) 4-12 for third-trimester equivalent rodents or PND 30-35 for adolescent/adult (mature) rodents. Pups

(PND 4-12) were housed with their dams, while mature rodents (PND 30-35) were housed separately according to sex. All animals were kept on a standard light/dark cycle with lights on at

07:00. Cerebellar slices of randomly selected male or female rodents were prepared each day of experimentation. Rodents were anesthetized with isofluorane and decapitated, with brain extraction occurring in 1°C aCSF containing 124 mM NaCl, 26 mM NaHCO3, 1 mM NaH2PO4, 2.5 mM KCl, 2.5 mM CaCl2, 2 mM MgCl2, and 10 mM D-glucose. All aCSF was bubbled with 95% O2/5%

CO2 and contained 1 mM kynurenic acid (a glutamate receptor antagonist used to prevent potential excitotoxicity within the tissue). The cerebellum was microdissected out and parasagittal slices (225 µm) of the cerebellar vermis were taken using a Leica VT1200S vibratome in a slicing chamber filled with bubbling 1°C aCSF. Slices were then incubated in aCSF containing

1 mM kynurenic acid at physiological temperature (34-35°C) for 1 hour before electrophysiological recordings were conducted.

Slice Electrophysiology. Slices were secured with a platinum harp in a submersion chamber mounted on an Olympus BX51WI microscope, and visualized with a 60X (0.90 N.A.) water- immersion objective. Slices were perfused at a rate of 5-7 ml/min with artificial cerebrospinal fluid (aCSF), maintained at a temperature between 32-36° C, and bubbled with a 95% O2/5% CO2 gas. Granule cells, Golgi cells, stellate/basket cells, and Purkinje cells were visually identified and voltage-clamped (Vh = -30mV; PND 19-36 Purkinje cell voltage was held at -60mV) with patch electrodes made from borosilicate glass capillary pipettes. Pipettes contained: 130 mM

CsGluconate, 4 mM NaCl, 0.5 mM CaCl2, 10 mM HEPES, 5 mM EGTA, 4 mM MgATP, 0.5 mM

Na2GTP, and 5 mM QX-314. Internal solution was pH-adjusted to 7.2-7.3 with CsOH. Electrode resistance was 4-8 MΩ for granule cells and stellate/basket cells, and 1-4 MΩ for Golgi and

Purkinje cells. Cells were excluded if access resistance changed by >20% throughout the duration

- of an experiment. Given the extra- and intracellular [Cl ], ECl was ~ -60mV, so voltage-clamping at

-30mV resulted in IPSCs being outward and EPSCs being inward. All recordings were filtered at

10 kHz and acquired at 20kHz. Mature Purkinje cells were voltage-clamped at the [Cl-] reversal potential (Vh=-60mV), electrically isolating EPSCs and allowing for their visualization. A glass capillary stimulating electrode was used to evoke action potentials in local afferents, with a stimulation range of 25µA-1.5mA, determined by an easily detectible and consistent amplitude evoked synaptic response. Drugs were dissolved in aCSF and constantly bubbled with 95% O2/5%

CO2 gas before being administered. All experimental conditions were conducted for 5 minutes each, with the baseline condition being conducted for at least 5 minutes or until a stable baseline was reached.

Analysis of spontaneous and evoked EPSC/IPSC currents. Spontaneous synaptic events (sIPSCs

& sEPSCs) from the final two minutes of recording in each experimental condition were analyzed using Mini Analysis 6.0.7 (Synaptosoft). Automatic detection of sIPSCs and sEPSCs was executed, using an amplitude threshold of 2 times the peak to peak amplitude of the noise, and then events were individually inspected with a further inclusion criterion of having a rise time at least 3 times faster than the decay time. Average frequency was determined including temporally overlapping events, then all non-overlapping events were averaged to calculate mean amplitude. All cells with lower than 0.1 Hz spontaneous baseline frequency were excluded from analysis. Because the

CB1R agonist WIN 55,212-2 fails to wash out within a reasonable amount of time, the wash condition is excluded in the following results unless noted otherwise. Electrically evoked (1 stim/20 sec; 10 stim/2 min) synaptic currents were quantified using pClamp 10.4 (Clampfit; Axon

Instruments, Foster City, CA). The final ten evoked responses in every condition were averaged, and the mean peak amplitude and paired pulse ratio was calculated.

Statistics. Data are expressed as the means ± the standard deviation of the means (M ± SEM) for each condition. Within-cell spontaneous PSC frequencies and evoked PSC amplitudes before and after drug treatments were compared using paired students t-tests. If normality tests failed (non- parametric), Wilcoxon Signed Rank tests were performed instead of students t-tests. Significance was p<0.05 unless otherwise noted.

Reagents. Kynurenic acid (1mM; Abcam; ab120064) was added to aCSF solution during brain extraction. Either spontaneous PSC or evoked PSCs were isolated using either 10 µM GABAzine

(Abcam; ab120042) (to isolate EPSCs) or 50µM AP5/25µM NBQX (Abcam; ab120003 & ab120046)

(to isolate IPSCs). To isolate mSPCs, 500nM TTX (Alomone Labs; T-550) was used. The CB1 agonist

(+)-WIN 55,212-2 was purchased from Cayman Chemical (Cat. No. 10009023) and CB1 inverse agonist SR141716A (Rimonabant) was purchased from Tocris (Cat. No. 0923). Primary antibodies were (host/supplier and catalog number/dilution): CB1R (rabbit/Synaptic Systems 258

003/1:1000), GAD 65 (mouse/EMD Millipore MAB351/1:500), GAD 67 (mouse/EMD Millipore

MAB5406/1:500), Calbindin (goat/Santa Cruz Biotech SC7691/1:500), VGluT 1/2

(chicken/Synaptic Systems 297104/1:1000) and GFAP (chicken/Synaptic Systems 173 006/1:500).

RESULTS

Immunohistochemistry

Immunohistochemistry was used as a simple method to gain spatial specificity of CB1R localization within the cerebellar cortex. Although lacking functional relativity, immunohistochemistry can be useful to observe general cerebellar cortex morphology and localized CB1R expression at various developmental time points. Immunohistochemistry can distinguish CB1Rs on specific cell types, which can then guide further electrophysiological experiments to confirm functional expression. Here we attempt to provide a general characterization of both spatial and temporal CB1R expression patterns of the cerebellar cortex, which may provide insight into its functional roles.

Adult/mature cerebellar cortex

Adult rat cerebellar tissue (PND 32) followed a similar pattern to what has been seen in previous literature157, with heavy CB1R expression within the molecular layer of the cerebellar cortex, and expression on what appears to be basket cell terminals onto Purkinje cells (PND 32) (Fig. 2.1A).

Expression within the molecular layer is thought to be located on granule cell parallel fibers, which are presynaptic to Purkinje cells, and has been confirmed through previous electrophysiological studies161–163. When comparing CB1R expression with the Purkinje cell marker Calbindin, CB1R expression appears to be absent from Purkinje cells, evident by the lack of CB1R florescence on Purkinje dendrites. Molecular layer interneuron somas appear to be void of strong CB1R expression, when compared with the surrounding molecular layer, although this may be overshadowed by surrounding fluorescence (Fig. 2.1A). There appears to be relatively less CB1R expression within the granule cell layer, but there are clusters of CB1Rs in what are

presumably glomeruli, the points of synaptic contact between mossy fibers and Golgi cell terminals with granule cell dendrites (Fig. 2.1B).

Developing cerebellar cortex

The cerebellar cortex undergoes many morphological changes as it develops. Comparing the thickness of the external granule layer (EGL) at different developmental time points shows a gradual shrinkage of the EGL as immature granule cells migrate away to the granule cell layer, and Purkinje cell dendrites proliferate to increase the size of the molecular layer. By PND 32, all granule cells within the EGL have migrated and Purkinje cell dendrites have reached their final morphological profile (Fig. 2.2A). Immunohistochemistry confocal imaging in PND 4 rat pups revealed a diffuse expression pattern of CB1Rs within the cerebellar cortex (Fig. 2.2B), with high levels of expression throughout the external granule layer (EGL), molecular layer, and granule cell layer. When comparing CB1R expression visually, this diffuse pattern of CB1R expression appears to become more focused with age, a progression seen when comparing PND 4 and 9 cerebellar tissue (Fig. 2.3). Although not quantified, CB1R expression in PND 4 tissue is quite diffuse but especially concentrated surrounding Purkinje cells and within granule cell layer glomeruli. At PND

9, CB1R expression is especially dense within the molecular layer, and appears to be more localized within the granule cell layer as opposed to PND 4 tissue (Fig. 2.3). Unlike mature tissue, there does not appear to be concentrated expression of CB1Rs on basket cells in developing tissue. In the granule cell layer, heavy CB1R expression appears to densely congregate near granule cell layer glomeruli (the point of synaptic contact between mossy fiber terminals, Golgi cell terminals, and granule cell dendrites) (Fig. 2.2C). There is clear colocalization between the

expression of CB1R and the glutamate vesicular transporter 1 & 2 (VGluT1/2), which labels glutamatergic afferents, in the absence of Hoechst (nuclear stain) (Fig. 2.2C). This colocalization may indicate expression of these proteins in the region between granule cell somas, and likely within glomeruli.

Because cannabinoid receptors, especially CB2Rs, are known to be expressed on glial cells, we used a glial cell marker (glial fibrillary acidic protein; GFAP) to determine whether glial expression could account for CB1R expression found within the molecular Layer. We used coronal cerebellar cortex slices from PND 10 rats in order to visualize parallel fibers as they pass along the molecular layer. Upon visual examination, we failed to observe clear localization between

CB1Rs and GFAP, indicating CB1R expression on glial cells is unlikely (Fig. 2.4B). This suggests

CB1R expression is expressed on neuronal fibers within the molecular layer.

Transgenic CB1R mice

To ensure our primary CB1R antibody binds specifically to CB1Rs, we compared CB1R expression between C57BL/6J wild-type and CB1R knock-out (KO) mice. There was a clear lack of CB1R expression in the cerebellar cortex of PND 9 CB1R KO mice (Fig. 2.5A) when compared with apparent expression levels in PND 10 WT mice (Fig. 2.5B). CB1R KO cerebellar tissue showed no clear concentration of staining as opposed to CB1R WT tissue, which clearly showed concentrated

CB1R expression within all layers of the cerebellar cortex. This was similar to expression seen in

PND 9 rats (compare to Fig. 2.3B), showing a continuity of expression across rodent species. In addition to developing tissue, CB1R expression is similar in mature mouse cerebellar tissue: there is ubiquitous CB1R expression in PND 106 CB1R WT mice (Fig. 2.5C), with heavy expression within

the molecular layer and on basket cells, but no clear CB1R expression in PND 90 CB1R KO mice

(Fig. 2.5D). The results of these immunohistochemical findings; specifically, the absence of CB1R fluorescence in transgenic mice lacking the CB1 gene, confirms that our primary CB1R antibody is specific to CB1R proteins. This gives credence to our previous immunohistochemical findings.

Patch-Clamp Electrophysiology

1. Granule cell synaptic input

Granule cells receive synaptic input from glutamatergic mossy fibers, whose cell bodies are located within the brainstem, and GABAergic Golgi cell interneurons, whose cell bodies also reside within the granule cell layer. Granule cell axons project up into the molecular layer (ML) to synapse onto Golgi, stellate, and basket, and Purkinje cells. However, little is known regarding

CB1R modulation of synaptic functionality on developing granule cell afferents. In this subsection we whole-cell patch clamp both developing and mature granule cells to determine functional

CB1R expression.

1.1 Developing tissue: Mossy fiber input to granule cells

When observing spontaneous excitatory post-synaptic currents (sEPSCs) in PND 4-12 rat whole- cell patch-clamped (Vh=-30mV) granule cells (Fig. 2.6A), bath application of the CB1R agonist WIN

55,212-2 (5µM) reduced sEPSC frequency (mean baseline frequency = 0.59 ± 0.21, % reduction =

63.8 ± 7.7, p<0.05, n=11) (Fig. 2.6B), agreeing with the canonical role CB1Rs play in reducing presynaptic vesicle release. The amplitude of sEPSCs did not change within the WIN condition

(16.3 ± 1.8 pA, % increase = 18.6 ± 15.3, p=0.31), indicating WIN effects likely had no influence on postsynaptic receptors.

When electrically stimulating mossy fibers in PND 8-12 rats (Fig. 2.6A), evoked EPSC

(eEPSC) amplitude (mean baseline amplitude = 61.3 ± 11.0 pA) is reduced (% reduction = 38.7 ±

7.8, p<0.01, n=16) following the application of WIN 55,212-2 (5µM) (Fig. 2.6C & D), likely a result of functional CB1R expression on mossy fiber terminals. Additionally, a second series of experiments was performed where eEPSCs were pharmacologically isolated using the GABAAR antagonist Gabazine. In the presence of Gabazine (10µM), eEPSC amplitude (mean baseline amplitude = 47.6 ± 16.5 pA) is reduced following WIN application (5µM) (% reduction = 40.7 ±

12.5, p<0.05, p<0.05, n=6) (Fig. 2.6E), confirming the effects of WIN are isolated to this specific synapse (not influenced by potential mossy fiber stimulation of Golgi cells).

To test whether the reduction in sEPSC frequency and eEPSC amplitude following WIN application is pre- or postsynaptically mediated, we performed paired-pulse ratio (PPR) experiments. We recorded from granule cells (Vh=-30mV) and electrically stimulated mossy fibers in quick succession, recording the ratio of eEPSC amplitudes before and after WIN application. Baseline PPR exhibited a paired-pulse depression (PPR = 0.77 ± 0.09) (Fig. 2.8A & B), but this switched to paired-pulse facilitation (PPR = 1.10 ± 0.16) following WIN (5µM) application

(% PPR increase = 43.6 ± 15.22, p<0.05, n=5) (Fig. 2.8B & C). A change in the PPR following WIN application supports presynaptic CB1R localization, and agrees with immunohistochemical data showing CB1R and VGluT1/2 colocalization within developing granule cell glomeruli (as seen in

Fig. 2.2C).

To more thoroughly characterize the location of CB1Rs in the mossy fiber -> granule cell synapse, recordings of quantal glutamatergic vesicle release were also performed in PND 4-9 granule cells by blocking action potential-induced vesicle release using the voltage-gated sodium channel blocker tetrodotoxin (TTX; 500nM) (Fig. 2.9). There was no reduction in EPSC event (now referred to as mini EPSCs (mEPSCs)) frequency following TTX (500nM) application alone (mean baseline frequency = 0.41 ± 0.11, n=17), suggesting that basal release is action potential- independent. However, WIN application (5µM) in the presence of TTX significantly reduced mEPSC frequency (mean TTX condition frequency = 0.32 ± 0.06, % reduction = 40.3 ± 6.4, p<0.01)

(Fig. 2.9A), yet mEPSC amplitude remained unchanged following TTX application (mean baseline amplitude = 17.5 ± 1.6 pA, % change = 0.4 ± 6.1, p=0.15) (Fig. 2.9B). A reduction in Ca2+-dependent vesicle release into the synapse in the absence of action potentials is expected if CB1Rs are located presynaptically, as the data implies. A lack of change in EPSC amplitude following TTX application may suggest glutamatergic vesicle release from mossy fibers is already quantal.

To ensure the effects of WIN 55,212-2 were solely due to CB1R activation, WIN (5µM) was bath applied to the slice in the presence of the CB1R inverse agonist SR141716A (1, 2, and 8µM in subsequent experiments). Previous literature has shown that 1µM SR141716A blocks the effects of 5µM WIN in PND 15-19 Purkinje cells163. In PND 3-10 rats, WIN (5µM) was still able to reduce granule cell sEPSC frequency in the presence of 1µM SR141716A (mean baseline frequency = 0.47 ± 0.12, % reduction = 41.1 ± 8.2, p<0.01, n=9). In a dose dependent manner, both 2µM and 8µM SR141716A blocked the WIN induced reduction in sEPSCs (2µM SR: mean baseline frequency = 0.53 ± 0.12, % reduction = 14.4 ± 19.2, p=0.32, n=8, Fig. 2.10A; 8µM SR: mean baseline frequency = 0.26 ± 0.07, % reduction = 7.5 ± 18.9, p=0.44, n=6) (Fig. 2.10B).

Additionally, we found that WIN (5µM) failed to reduce mossy fiber stimulated eEPSC amplitude

(mean baseline amplitude = 46.1 ± 17.2 pA; % change = 2.4 ± 4.6, p=0.55, n=10) in the presence of 1µM SR141716A when recording from granule cells (Fig. 2.10D). There may be potential mechanistic explanations for the discrepancy between 1µM SR141716A block in our spontaneous and evoked results (as addressed in the discussion). Regardless of mechanism, these data support the assumption that reduced mossy fiber glutamate release seen following application of CB1R agonist WIN 55,212-2 is solely acting on CB1Rs in these experiments.

1.2 Developing tissue: Golgi cell input to granule cells

Whole-cell patch-clamp recording (Vh=-30mV) from PND 4-12 rat granule cells revealed that CB1R agonist WIN 55,212-2 (5µM) reduces GABAergic Golgi cell sIPSC frequency (mean baseline frequency = 0.88 ± 0.17, % reduction = 43.4 ± 20.1, p<0.01, n=19) (Fig. 2.7A), suggesting CB1Rs are expressed on Golgi cell terminals. Similar to mossy fiber synaptic input, sIPSC amplitude was also unchanged following WIN application (15.2 ± 1.3 pA, % increase = 0.7 ± 5.8, p=0.94).

When locally stimulating an IPSC response through local Golgi cell electrical stimulation

(eIPSC), WIN 55,212-2 (5µM) application surprisingly failed to reduce eIPSC amplitude (mean baseline amplitude = 29.68 ± 5.56 pA; % change = 12.1 ± 21.6, p=0.55, n=11) (Fig. 2.7B). In order to explore this further, we blocked action potentials and isolated action potential independent

GABAergic vesicle release into the synapse using the voltage-gated sodium channel blocker tetrodotoxin (TTX; 500nM). In the presence of TTX, sIPSC frequency was reduced (mean baseline frequency = 0.54 ± 0.09, % reduction = 54.9 ± 10.5, p<0.01, n =27) (Fig. 2.9A), as well as sIPSC amplitude (mean baseline amplitude = 21.07 ± 2.32, % reduction = 28.1 ± 6.4, p<0.01) (Fig. 2.9B).

A reduction in IPSC frequency following TTX application suggests some sIPSCs under control conditions were action potential-dependent, and reduced amplitude implies action potential- dependent events were multi-quantal. WIN 55,212-2 (5µM) application in the presence of TTX

(500nm) significantly reduced mIPSC frequency (mean baseline TTX frequency = 0.19 ± 0.04, % reduction = 30.9 ± 7.9, p<0.05) (Fig. 2.9A), but not amplitude (% reduction = 4.1 ± 4.0, p=0.22).

Differing mechanistic effects of CB1R activation, such as effects on voltage-gated calcium channels discussed in the Discussion section, may explain the discrepancy between a lack of eIPSC amplitude reduction but reduced mIPSC frequency in the presence of WIN.

1.3 Mature granule cells

Interestingly, whole-cell patch-clamp of PND 19-36 granule cells (Vh=-30mV) revealed that CB1R agonist WIN 55,212-2 (5µM) continued to reduce GABAergic Golgi cell sIPSC frequency (mean baseline frequency = 0.39 ± 0.06; % reduction = 46.2 ± 13.8; p<0.01, n=27) (Fig. 2.11B), but no longer reduced glutamatergic mossy fiber sEPSC frequency (mean baseline frequency = 0.26 ±

0.03, p>0.05, n=16) (Fig. 2.11A). Spontaneous amplitudes were unchanged for sEPSCs following drug application (mean baseline amplitude = 14.9 ± 0.9 pA, % change = 5.6 ± 3.2, p=0.13), but were significantly reduced for sIPSCs (mean baseline amplitude = 8.3 ± 0.5 pA (% reduction = 8.6

± 3.4, p<0.05, Fig. 2.11C). This may indicate the reduction in sIPSC frequency lowers synaptic

GABA concentrations below saturating levels, causing a slight reduction in amplitude. The lack of

WIN effect at the mossy fiber to granule cell synapse may imply there is developmentally restricted CB1R expression since we see a strong WIN effect at this synapse within young tissue.

1.4 CB1R KO/WT mice

In addition to using transgenic mice for prior immunohistochemical comparisons, granule cell synaptic activity in developing and mature CB1R knockout (KO) and wild type (WT) mice were compared using whole-cell patch-clamp recordings (Vh=-30mV). In CB1R WT mice, granule cells in developing (PND 6-10) tissue showed a reduction in both glutamatergic mossy fiber sEPSC frequency (mean baseline frequency = 0.38 ± 0.08; % reduction = 27.3 ± 11.2%, p<0.05, n=16)

(Fig. 2.12A) and GABAergic Golgi cell sIPSC frequency (mean baseline frequency = 0.35 ± 0.07; % reduction = 34.2 ± 7.4%, p<0.05, n=10) (Fig. 2.12C) following WIN (5µM) application. Granule cells in mature (PND 30-34) tissue showed no change in sEPSC frequency following WIN (5µM) application (mean baseline frequency = 0.28 ± 0.06; % change = 2.0 ± 9.0, p>0.05, n=15) (Fig.

2.13B), but there was a reduction in sIPSC frequency (mean baseline frequency = 0.32 ± 0.09; % reduction = 42.1 ± 8.8%, p<0.05, n=9) (Fig. 2.13D). These data agree with our age-dependent granule cell data in rats. In both developing (PND 7-9) and mature (PND 31-35) CB1R KO mice, granule cell sEPSC and sIPSC baseline frequencies (PND 7-9: 0.54 ± 0.12 (n=12) (Fig. 2.13A) & 0.37

± 0.09 (n=9) (Fig. 2.13C); PND 31-35: 0.34 ± 0.03 (n=9) (Fig. 2.13B) & 0.31 ± 0.07 (n=6) (Fig. 2.13D), respectively) were unchanged following CB1R agonist WIN 55,212-2 (5µM) application, which supports our previous findings demonstrating WIN effects are on CB1Rs here. Amplitudes were unchanged following drug application for all WT and KO groups at both ages: (PND 6-10 (mean baseline amplitude (pA) = (WT sEPSCs) 15.1 ± 1.5, p=0.24; (KO sEPSCs) 13.9 ± 1.4, p=0.12; (WT sIPSCs) 10.0 ± 1.6, p=0.24; (KO sIPSCs) 8.8 ± 0.9, p=0.33) and (PND 30-34 (mean baseline amplitude (pA) = (WT sEPSCs) 10.8 ± 0.7, p=0.95; (KO sEPSCs) 13.9 ± 1.4, p=0.27; (WT sIPSCs) 8.6

± 1.1, p=0.31; (KO sIPSCs) 7.0 ± 0.9, p=0.98) (Table 2.1).

1.5 Reconciling the prolonged WIN effect in Wash condition

The CB1R agonist WIN 55,212-2 is notorious for failing to wash out of tissue do to its hydrophobicity. Consequentially, it is difficult to determine precisely why there is a prolonged

WIN effect (decrease in sPSC frequency) during the Wash condition. For example, is the inability to rebound to baseline spontaneous frequency due to “sticky” WIN, or could there be a prolonged intracellular effect in the absence of the drug following Wash? In order to parcel out these distinct possibilities, we recorded from PND 3-7 rat granule cells (Vh=-30mV) and applied the CB1R inverse agonist SR141716A (1µM) in the presence of WIN (5µM) to determine whether this could return sPSC frequency back to baseline. When quantifying sEPSCs (mean baseline frequency = 0.24 ± 0.05), WIN (5µM) application reduced the frequency as expected (% reduction from baseline = 66.6 ± 8.5, p<0.01, n = 12), and the subsequent addition of SR141716A (1µM) in the presence of WIN significantly increased sEPSC frequency back to baseline levels (% increase from WIN = 106.9, p<0.01). The subsequent condition absent of SR141716A (still in WIN; SR

“wash”) again decreased sEPSC frequency (% decrease from WIN+SR = 21.6 ± 14.0, p<0.05) (Fig.

2.13A). There were no differences in sEPSC amplitude following drug application (mean baseline amplitude = 14.9 ± 2.1 pA, % change = 6.8 ± 14.6, p=0.62), which may indicate no postsynaptic modifications.

Similarly, when quantifying sIPSCs (mean baseline frequency = 0.67 ± 0.18), WIN (5µM) application reduced the frequency (% reduction from baseline = 30.0 ± 12.2, p<0.05, n = 12), but again, the addition of SR141716A (1µM) in the presence of WIN returned sEPSC frequency back to baseline levels (% increase from WIN = 61.1 ± 35.6, p<0.05). However, sIPSC frequency did not

change following removal of SR141716A in the presence of WIN (% decrease from WIN+SR = 10.2

± 11.6, p>0.05). This is unlike sEPSCs, which did reduce in frequency following SR141716A removal (Fig. 2.13B), which may imply the inverse agonist SR141716A has prolonged an intracellular effect in Golgi presynaptic terminals at the Golgi->granule synapse. Again, there were no differences in sIPSC amplitude following drug application (mean baseline amplitude =

16.0 ± 1.7 pA, % change = 11.8 ± 11.5, p=0.40), suggesting observed effects were likely presynaptic.

2. Purkinje cell synaptic input

Most previous research regarding the cerebellar endocannabinoid system has been focused on mature Purkinje cell synapses. CB1Rs have been characterized on glutamatergic parallel fibers and GABAergic stellate and basket cell terminals onto Purkinje cells162,163. Endogenous endocannabinoid release has also been studied at these synapses, exhibiting depolarization- induced suppression of excitation and inhibition (DSE and DSI)133,134,225. What is not known is whether CB1Rs are developmentally expressed in these synapses, and whether they function similarly in developing tissue. Thus, we attempted to expand on previous findings in the mature cerebellum to examine the role of CB1Rs on developing Purkinje cell signaling.

2.1 Adult (PND 19-36) eEPSCs

In whole-cell patch-clamped rat Purkinje cells (Vh=-60mV), parallel fiber-evoked eEPSCs were analyzed (Fig. 2.14A) in an attempt to replicate previous findings. WIN 55,212-2 (5µM) administration significantly reduced eEPSC amplitude (mean baseline amplitude = 80.2 ± 13.3 pA,

% reduction = 40.8 ± 9.2, p<0.01, n=18) (Fig. 2.14B & C). This agrees with previous observations finding parallel fiber terminal CB1R activation strongly reduces glutamatergic vesicle release.

Surprisingly, the eEPSC paired pulse ratio (PPR) was decreased following WIN application (mean baseline PPR = 2.92 ± 0.16, % reduction = 11.75 ± 4.80, p<0.05) (Fig. 2.14D). A change in PPR suggests CB1R activation has a presynaptic mechanism of action, but a decreased PPR is counter to what would be expected from CB1R activation, which reduces vesicle release probability. It is also counter to what is reported in Purkinje cells in previous literature107,166,226.

2.2 Developing tissue: Parallel fiber input

When whole-cell patch clamping Purkinje cells in PND 4-10 rats (Vh=-30mV) (Fig. 2.15A), bath application of WIN 55,212-2 (5µM) failed to alter either glutamatergic parallel fiber sEPSC frequency (mean baseline frequency = 1.32 ± 0.28, % change = 17.3 ± 14.1, p=0.36, n=30) (Fig.

2.15B) or locally evoked parallel fiber eEPSC amplitude (mean baseline amplitude = 316.91 ±

97.46 pA, % change = 19.0 ± 3.0 p=0.10, n=13) (Fig. 2.14C). The sEPSC amplitude was unchanged

(mean baseline amplitude = 22.6 ± 4.0 pA, % change = 3.3 ± 4.5, p=0.33) following WIN application

(Fig. 2.16). These data are opposed to the results of previous studies163 and our own findings in older tissue (Fig. 2.14) showing a strong reduction in eEPSC amplitude in Purkinje cells. This lack of effect may indicate parallel fiber terminals onto Purkinje cells may not express CB1Rs, or at least functional CB1Rs, during this particular (PND 4-10) developmental period.

2.3 Developing tissue: Stellate/Basket cell input

In Purkinje cells (Vh=-30mV), bath application of WIN 55,212-2 (5µM) failed to alter either

GABAergic sIPSC frequency (mean baseline frequency = 1.28 ± 0.29, p>0.05, n = 21) (Fig. 2.15B) or local molecular layer interneuron evoked eIPSC amplitudes (mean baseline amplitude = 164.24

± 40.32 pA, % change = 3.3 ± 3.5, p=0.95, n=4) (Fig. 2.15D). The sIPSC amplitudes were unchanged

(mean baseline amplitude = 60.18 ± 24.59 pA, % change = 9.0 ± 4.0, p=0.18) following WIN application (Fig. 2.16), implying no postsynaptic modifications by WIN. These results were similar to the lack of WIN effect in parallel fiber synapses, suggesting a lack of functional CB1R expression on stellate and basket cell terminals onto Purkinje cells.

2.4 Climbing fiber input

While we did not record Purkinje cell responses to climbing fiber (CF) activity, previous studies have found that 1) WIN (5µM) greatly reduces CF-stimulated glutamatergic eEPSC amplitude in

PND 16 rats163, and 2) Purkinje cell mGluR1 receptor activation in PND 8-13 mice stimulates endocannabinoid production that reduce CF-stimulated neurotransmitter release, which is blocked with a CB1R antagonist178. These studies provide evidence of functional CB1R expression on climbing fibers during development, although the time of onset remains to be determined.

2.5 CB1R WT mice

To determine the robustness of the CB1R agonist WIN lack of effect on developing Purkinje cell afferents, we whole-cell patch-clamped Purkinje cells (Vh=-30mV) in developing (PND 8-9) CB1R

WT mice. There was no change in either sEPSC or sIPSC frequency (mean baseline frequency =

5.44 ± 2.20 and 4.0 ± 0.58, respectively) following WIN 55,212-2 (5µM) application (p>0.05, n=9,

each) (Fig. 2.17). The amplitude of both sEPSCs and sIPSCs (mean baseline amplitude (pA) = 24.2

± 2.08 (% change = 0.2 ± 4.3, p=0.78) also remained unchanged. The lack of spontaneous frequency change following WIN application in wildtype mice agrees with our data showing a lack of WIN response in PND 4-10 rat Purkinje cells.

2.6 Gabazine + CB1R inverse agonist SR sEPSCs

There is a possibility that the CB1R agonist WIN has no effect on Purkinje afferent vesicle release due to tonic endogenous CB1R activation. Therefore, we attempted to determine whether blocking potential CB1Rs on Purkinje cell afferents could increase neurotransmitter release onto rat Purkinje cells. PND 8-10 Purkinje whole-cell patch clamp recordings (Vh=-60mV) were conducted in the presence of Gabazine and the CB1R inverse agonist SR141716A. Bath application of SR141716A (2µM) in the presence of Gabazine (10µM) increased sEPSC frequency

(mean baseline frequency = 10.32 ± 1.84, % increase = 24.85 ± 9.37, p<0.05, n = 14) (Fig. 2.18), but sEPSC amplitude was unaffected by SR141716A (mean baseline amplitude = 20.2 ± 1.5 pA, % change = 6.7 ± 5.8, p=0.27). These results are surprising given the CB1R agonist WIN 55,212-2 failed to change Purkinje cell sEPSC frequency. An increase in sEPSC frequency following application of CB1R inverse agonist SR141716A may suggest either 1) a tonic endocannabinoid presence that is normally suppressing sEPSC frequency, or 2) SR141716A may be actively suppressing, rather than simply blocking, CB1R activity presynaptically and further lessening

CB1R baseline inhibition of vesicle release mechanisms.

3. Stellate/basket cell synaptic input

Stellate and basket cell GABAergic interneurons are found within the molecular layer of the cerebellar cortex, and are thought to provide inhibition to laterally located Purkinje cells following excitation from granule cell parallel fibers. However, unlike Golgi cell GABAergic interneurons, stellate and basket cells have direct inhibitory contact onto Purkinje cells themselves. Because stellate and basket cells are often difficult to distinguish based on their location and morphology, we have grouped the two cell types together. Following florescent cell- filling, we were able to morphologically distinguish some Stellate and basket cells (Fig. 2.19B), but the majority were either difficult to parcel out, or were not cell-filled.

3.1 Developing tissue: Parallel fiber input

When whole-cell patch-clamp recording from PND 8-10 stellate and basket cells (Vh=-30mV), bath application of WIN 55,212-2 (5µM) failed to reduce either sEPSC frequency (mean baseline frequency = 0.59 ± 0.11, p>0.05, n=24) (Fig. 2.19C & D) or sEPSC amplitude (mean baseline amplitude = 30.96 ± 2.49 pA, % change = 3.4 ± 5.2, p=0.88). This lack of WIN effect suggests an absence of functional CB1R expression at these particular parallel fiber synapses.

3.2 Developing tissue: Stellate/basket input

Unlike sEPSCs, bath application of WIN 55,212-2 (5µM) when recording from PND 8-10 stellate and basket cells (Vh=-30mV) reduced sIPSC frequency (mean baseline frequency = 0.55 ± 0.13, % reduction = 28.3 ± 10.0, p<0.05, n = 15) (Fig. 2.19C & D), although there were no changes in sIPSC amplitude following WIN application (mean baseline amplitude = 17.45 ± 1.94 pA, % change = 3.0

± 4.9, p=0.99). A reduction in sIPSC frequency following WIN application may provide evidence

that CB1Rs are expressed on GABAergic stellate and basket cell afferents. It is likely these inputs are from fellow stellate and basket cells, although further experiments may need to be conducted to determine this definitively.

4. Golgi synaptic input

GABAergic Golgi cell interneurons are located within the granule cell layer of the cerebellar cortex, but their dendrites extend up into the molecular layer to form synapses with parallel fibers. Counter to intuition based on morphology and the location of Golgi cell dendrites, there has been recent compelling evidence suggests Golgi cells only receive GABAergic innervation from fellow Golgi cells, rather than stellate and basket cells located within the molecular layer26.

Golgi cell axons generally terminate onto granule cells, and release GABA, which binds to

GABAARs on granule cell dendrites. There is little to no information regarding Golgi cell CB1R expression, and it is currently unknown if the endocannabinoid system is involved in its synaptic modulation.

4.1 Parallel fiber input

When whole-cell patch clamping Golgi cells (Vh=-30mV) in PND 9-10 rats, bath application of WIN

55,212-2 (5µM) led to a reduction in glutamatergic parallel fiber sEPSC frequency (mean baseline frequency = 2.25 ± 0.33, % reduction = 37.0 ± 6.5, p<0.01, n = 22) (Fig. 2.20C & D). The sEPSC amplitude was unchanged following WIN application (mean baseline amplitude = 20.02 ± 1.25 pA, % change = 3.0 ± 4.2, p=0.25). A reduction in sEPSC frequency suggests there may be CB1R activation on granule cell parallel fiber terminals onto Golgi dendrites within the molecular layer.

This result is interesting as CB1R expression on these parallel fiber synapses may provide a partial explanation for our immunohistochemical results finding CB1R expression within the molecular layer.

4.2 Golgi cell input

When whole-cell patch clamping Golgi cells (Vh=-30mV) in PND 9-10 rats, bath application of WIN

55,212-2 (5µM) also reduced sIPSC frequency (mean baseline frequency = 1.74 ± 0.52, % reduction = 45.1 ± 7.1, p<0.01, n = 14) (Fig. 2.20C & D), but sIPSC amplitude was unchanged

(mean baseline amplitude = 12.55 ± 1.05 pA, % change = 4.1 ± 4.1, p=0.32). These GABAergic synaptic events onto Golgi cells likely originate from fellow Golgi cells26, and may also partially explain molecular layer CB1R expression since Golgi cell dendrites extend into the molecular layer.

FIGURE 2.1

Figure 2.1. CB1R expression within the mature (PND 32) A cerebellar cortex. (A) Immunohistochemistry of PND 32 rat cerebellar cortex showing heavy CB1R localization in the molecular layer (ML), on basket cell-Purkinje cell synapses (see asterisks), and within presumed glomeruli within the granule cell layer (GL). Calbindin (cyan) is a 2+ Ca sequestering protein that indiscriminately labels Purkinje cells within the cerebellar cortex. Hoechst (magenta) is a nuclear stain, showing dense GCs within the GCL, with sparse interneurons within the ML. (B) A zoomed image (63X) of PND 32 granule cell layer glomeruli B showing CB1R expression (green) and GABAergic terminal markers GAD 65 & 67 (red) located in the space void of cell nuclei (magenta; Hoechst). This correlates with the region of 5µm 5µm synaptic connectivity (glomeruli) CB1R GAD 65/67 between glutamatergic Mossy Fiber and GABAergic Golgi cell terminals with granule cell dendrites, suggesting CB1Rs may be found on these terminals. ML=Molecular Layer; PL=Purkinje Layer; GL=Granule Layer

5µm 5µm Hoechst Merge

FIGURE 2.2 A B

Figure 2.2. Developmental CB1R expression within the developing (PND 4-12) cerebellar cortex. (A) Schematic depicting the development of the cerebellar cortex, showing immature granule cells (magenta) migrating from the external granule layer (EGL) to the granule cell layer (GL) concurrently with the proliferation of Purkinje dendritic trees (cyan). Granule cells have finished migrating by PND 32, seen by the diminishment of the EGL and proliferation and full development of Purkinje dendritic trees within the molecular layer. (B) Immunohistochemistry showing spatial expression of CB1Rs in PND 4 rat cerebellar cortex. CB1R expression appears more diffuse and less localized than mature cerebellar cortex, with heavy expression within the granule layer and localized near Purkinje cell somas. (C) A zoomed image (63X) of CB1R localization within the granule cell layer. Arrows point to regions of colocalization between CB1Rs and glutamatergic synaptic markers VGluT 1 & 2, in the absence of granule cell nuclei, suggesting expression within granule cell layer glomeruli. EGL=External Granule Layer; ML=Molecular Layer; PL=Purkinje Layer; GL=Granule Layer

FIGURE 2.3

Figure 2.3. Progression of CB1R expression during

A development of the rat cerebellar cortex. (A) PND 4 rat cerebellar cortex showing diffuse CB1R expression (green) throughout all layers of the cerebellar cortex, with markedly dense expression concentrated around the Purkinje and molecular layers, but also within the extracellular milieu of the granule cell layer. (B) PND 9 rat cerebellar cortex showing CB1R expression (green) becoming denser within the molecular layer as it grows in thickness, but is still found within the Purkinje and granule cell layers. Green: B CB1Rs; Cyan: Calbindin, which specifically labels Purkinje cells; Magenta: Hoechst, which labels cell nuclei.

FIGURE 2.4

25µM CB1R 25µM GAD 65/67

25µM GFAP 25µM Merge

Figure 2.4. Coronal section of cerebellar cortex showing CB1R and glial expression in PND 7 rat. (A) Schematic contrasting the orientation between Purkinje cell dendritic trees and parallel fibers in sagittal versus coronal cerebellar sections. Coronal sections were taken in order to visualize parallel fibers lengthwise. (B) Parallel fibers display heavy CB1R expression (green) within the molecular layer, which does not appear to colocalize with the glial cell marker GFAP (red). GABAergic markers GAD 65 & 67 were used in order to visualize the location of GABAergic terminals and Purkinje cells.

FIGURE 2.5 A B

C D

Figure 2.5. CB1R immunohistochemistry antibodies appear CB1R-specific when comparing transgenic CB1R knockout and wildtype mice. (A) CB1R staining in PND 10 wildtype (WT) mouse shows similar expression to that found within PND 9 rats, with heavy labeling in the molecular layer, heavily dispersed throughout the granule cell layer, and potentially within the external granule layer. (B) CB1R staining in PND 9 knockout (KO) mouse shows light background staining of the tissue, indicating the antibody fails to discriminately bind within the tissue, and verifying CB1R specificity of the immunohistochemistry in CB1R WT mice. (C) CB1R specificity in PND 106 WT mouse, which displays similar specificity as found in PND 32 rats (see Fig. 2.1A), with heavy expression within the molecular layer and on basket cell terminals onto Purkinje cells. (D) CB1R staining in PND 90 KO mouse shows light and diffuse CB1R background florescence within the tissue, indicating the CB1R immunohistochemistry, when compared with WT tissue, is specific for CB1Rs.

FIGURE 2.6 A B

C D

Figure 2.6. CB1R agonist WIN 55,212-2 (5µm) reduces PND 4-12 granule cell sEPSC frequency and mossy fiber-evoked EPSC amplitude. (A) Circuit diagram showing whole-cell patch recording of granule cells (Vh = -30mV) while stimulating mossy fiber afferents. (B) An inset representative trace showing granule cell sEPSC frequency was reduced following 5µM WIN application, which is quantified in the corresponding sEPSC frequency graph. (C) A representative trace showing mossy fiber eEPSCs are reduced in amplitude following WIN application, which is quantified in (D), showing mean eEPSC amplitudes. (E) Granule cell eEPSC amplitude was also reduced in the presence of the GABAAR antagonist Gabazine (10µM), indicating the reduction in glutamatergic vesicle release is not influenced by GABAergic synaptic transmission. * indicates p<0.05; ** indicates p<0.01

FIGURE 2.7 A B

C D

Baseline 5µM WIN

Figure 2.7. CB1R agonist WIN 55,212-2 reduces sIPSC frequency in PND 4-12 granule cells, but fails to reduce eIPSC amplitude. (A) A circuit diagram depicting a whole-cell voltage

clamped (Vh=-30mV) granule cell while locally stimulating GABAergic Golgi cells. (B) Inset representative traces showing a reduction in Golgi cell GABAergic vesicle release onto granule cells following WIN (5µM) application, which is quantified as the overall mean sIPSC frequency in the corresponding graph. (C) Representative traces showing granule cell eIPSCs when locally stimulating Golgi cells; 5µM WIN application fails to reduce eIPSC amplitude, as quantified in (D), which displays the mean eIPSC amplitude. ** indicates p<0.01

FIGURE 2.8 A

B C

Figure 2.8. CB1R agonist WIN 55,212-2 increases mossy fiber-stimulated eEPSC Paired Pulse Ratio (PPR) in PND 4-10 granule cells, indicating CB1R-mediated presynaptic modulation. A) Representative traces showing mossy fiber paired pulse stimulated eEPSCs while recording from granule cells. The ratio (Pulse 2/Pulse 1) between eEPSC amplitudes following WIN (5µM) application increases between stimulations. B) A scaled overlay of both traces in each condition, displaying the PPR percentage change following WIN application. The PPR increases by 35% in the WIN condition. C) The quantification of mean PPR values show an overall increase in PPR following WIN application. The PPR, which exhibited Paired Pulse Depression in baseline conditions, switched to Paired Pulse Facilitation following application of CB1R agonist WIN 55,212-2. This indicates that CB1R activation reduces vesicle release probability, which enables a greater proportion of vesicles in the readily releasable pool to be released following the second pulse. Overall, the change in PPR indicates that the effects of WIN in the Mossy fiber->granule cell synapse are presynaptic in nature.

FIGURE 2.9 A

Figure 2.9. CB1R agonist WIN reduces mEPSC and mIPSC frequency in PND 4-9 granule cells. (A) TTX (500nM) application reduces sIPSC frequency, but not sEPSC frequency, suggesting sEPSCs are action potential-independent. Both mEPSC and mIPSC frequencies following bath application of 500nM TTX + 5µM WIN were reduced, which suggests CB1R activation acts presynaptically at both mossy fibers and golgi cell terminals. (B) sIPSC, but not sEPSC, amplitude is reduced following 500nM TTX application, which may indicate Golgi vesicle release is partially action potential dependent, and quantal size is reduced. * indicates p<0.05; ** indicates p<0.01

FIGURE 2.10 A B

2µM SR 2µM SR + 5µM WIN

C D

2µM SR 2µM SR + 5µM WIN

Figure 2.10. Actions of CB1R agonist WIN 55,212-2 are blocked using the CB1R-specific inverse agonist SR141716A in PND 3-10 granule cells. (A) Inset granule cell sEPSC representative traces showing CB1R inverse agonist SR141716A (2µM) blocked WIN-induced reduction in granule cell sEPSC frequency, showing (85.6% ±19.2) block, as depicted in the graph showing mean frequency. (B) Actions of WIN 55,212-2 (5µM) were blocked by SR141716A (SR) in a dose dependent manner when observing the percent change of sEPSC frequency from baseline. The percent frequency change in 1µM SR was significantly greater than baseline, indicating WIN was still able to reduce sEPSC frequency. Actions of WIN were blocked in 2µM and 8µM SR. (C) A representative trace showing 2µM SR blocked WIN-indced reduction in granule cell sIPSC frequency. All doses of CB1R inverse agonist SR141716A prevented a reduction in sIPSC frequency following the application of WIN 55,212-2 (5µM), as quantified in the graph. (D) Representative trace showing SR (1µM) blocked WIN-induced reduction in mossy fiber stimulated eEPSC amplitude. * indicates p<0.05; ** indicates p<0.01

FIGURE 2.11 A

B C

Figure 2.11. CB1R agonist WIN 55,212-2 fails to reduce granule cell sEPSC frequency in mature (PND 29-36) granule cells, but continues to reduce sIPSC frequency. (A) WIN 55,212- 2 (5µM) failed to reduce granule cell sEPSC frequency in granule cells (see representative trace inserts; downward deflections), in contrast to developing (PND 4-10) tissue, suggesting developmentally restricted expression of CB1Rs on mossy fiber terminals. (B) WIN 55,212-2 (5µM) continues to reduce granule cell sIPSC frequency in mature rat PND 29-36 granule cells (see representative trace inserts, upward deflections), suggesting a continued role of CB1R expression on Golgi cell terminals. (C) WIN 55,212-2 (5µM) also reduces granule cell sIPSC amplitude. ** indicates p<0.01

FIGURE 2.12 A B

C D

Figure 2.12. CB1R agonist WIN 55,212-2 fails to alter synaptic vesicle release in CB1R knock out mice in developing and mature granule cells. (A) In PND 7-9 mice, WIN 55,212-2 (5µM) reduced granule cell sEPSC frequency in CB1R wildtype (WT) mice, but not CB1R knockout (KO) mice (see inset representative traces). (B) PND 30-35 granule cell sEPSC frequency is unchanged by WIN 55,212-2 (5µM) in both CB1R KO and WT mice. (C) PND 7-9 sIPSC frequency is reduced in CB1R WT, but not CB1R KO mice. (D) PND 30-35 sIPSC frequency is reduced in PND 30-35 granule cells, which is also observed in mature rat granule cell tissue. These results confirm CB1Rs are functionally ablated from transgenic CB1R knockout mice. * indicates p<0.05

FIGURE 2.13 A

Figure 2.13. In PND 3-7 granule cells, CB1R inverse agonist SR141716A rescues CB1R agonist WIN-induced reduction in sPSC frequency. (A) The initial WIN condition reduced granule cell sEPSC frequency, with the following WIN (5µM) + SR (1µM) condition significantly increasing sEPSC frequency from the WIN condition, and was not significantly different from baseline. The final 5µM WIN “wash” condition again reduced sEPSC frequency, and was significantly lower than baseline. (B) WIN (5µM) reduced granule cell sIPSC frequency, and subsequent application of WIN (5µM) + SR (1µM) returned sIPSC frequency to baseline frequency. The following 5µM WIN “wash” failed to reduce sIPSC frequency. * indicates p<0.05; ** indicates p<0.01

FIGURE 2.14 A B

C D

Figure 2.14. CB1R agonist WIN 55,212-2 reduces Purkinje cell parallel fiber-stimulated eEPSC amplitude and paired pulse ratio (PPR) in the mature (PND 29-36) cerebellar cortex.

(A) A circuit diagram depicting voltage-clamped recordings (Vh = -60mV) of Purkinje cells in mature PND 29-36 rats while locally stimulating granule cell parallel fibers. (B) Representative traces depicting Purkinje cell eEPSCs from paired-pulse locally stimulating glutamatergic granule cell parallel fibers. Application of CB1R agonist WIN (5µM) dramatically reduced eEPSC amplitude in both first and second pulses, as quantified in (C), showing the mean eEPSC amplitude reduction in both pulses. (D) The Paired Pulse Ratio (PPR) between the two pulse stimulations were quantified, and found to be significantly reduced following WIN 55,212-2 (5µM) application. In each condition, the PPR shows Paired Pulse Facilitation, indicating a generally low probability of release at the granule cell- >Purkinje cell synapse. * indicates p<0.05; ** indicates p<0.01.

FIGURE 2.15 A B

C D

Figure 2.15. CB1R agonist WIN55,212-2 fails to reduce PND 4-10 Purkinje cell sPSC frequency or ePSC amplitude. (A) Circuit diagram showing whole-cell patch recording of Purkinje cells (Vh = -30mV) while locally stimulating either glutamatergic parallel fibers or GABAergic stellate/basket cell PC afferents. (B) WIN (5µM) fails to reduce both parallel fiber sEPSC and stellate/basket cell sIPSC frequencies, (see inset representative traces (sEPSCs p=0.91; sIPSCs p=0.46). (C) WIN (5µM) failed to reduce eEPSC amplitude following local stimulation of glutamatergic parallel fibers (p=0.10) (see inset representative traces), with mean eEPSC amplitude quantified in the corresponding graph. (D) WIN also failed to reduce eIPSC amplitude following local stimulation of GABAergic stellate and basket cells (p=0.95) (see inset representative traces), with mean eIPSC amplitude quantified in the corresponding graph.

FIGURE 2.16

Figure 2.16. CB1R agonist WIN 55,212-2 fails to alter PND 4-10 Purkinje cell sPSC amplitude. The mean baseline amplitudes of PND 4-10 Purkinje cell sEPSCs and sIPSCs did not change following bath application of CB1R agonist WIN 55,212-2 (5µM). This indicates there were no postsynaptic actions of WIN.

FIGURE 2.17

A B

Figure 2.17. CB1R agonist WIN 55,212-2 fails to alter Purkinje cell sPSC frequency in PND 8- 9 CB1R WT mice. The mean baseline frequency of PND 4-10 mouse Purkinje cell sEPSCs and sIPSCs did not change following bath application of CB1R agonist WIN 55,212-2 (5µM). The lack of WIN effect on spontaneous frequency suggests a lack of functional CB1Rs on Purkinje cells afferents and agrees with our developmental rat data.

FIGURE 2.18

Figure 2.18. CB1R inverse agonist SR141716A increases sEPSC frequency in PND 8-10 Purkinje cells. In the presence of the GABAAR Gabazine (10µM), application of CB1R inverse agonist SR141716A (2µM) slightly increased Purkinje cell sEPSC frequency. Spontaneous frequency did not return to baseline levels following second Gabazine condition. An increase in sEPSC frequency may have several implications, including endogenous tonic parallel fiber CB1R activation, or a result of intracellular mechanisms triggered by an inverse agonist. * indicates p<0.05

FIGURE 2.19 A B GC

GC L C D

Figure 2.19. CB1R agonist WIN 55,212-2 reduces sIPSC frequency, but not sEPSC frequency, in PND 8-10 stellate and basket cells. (A) Circuit diagram depicting a whole-cell patch recording from stellate and basket cells (Vh = -30mV). (B) Examples of stellate and basket cells that were visually identified using both DIC and fluorescent imaging. All stellate and basket cells were located within the molecular layer (ML), which is separated from the granule cell layer (GCL) by the Purkinje layer (Purkinje cells labeled by asterisks). (C) Representative traces showing stellate/basket cell sEPSC frequency (inward) was unchanged following 5µM WIN application, whereas sIPSC frequency (outward) was reduced, as quantified in (D), where mean frequencies were quantified. Reduced sIPSC frequency may indicate functional CB1R expression on GABAergic afferents to stellate and basket cells. * indicates p<0.05

FIGURE 2.20 A B

Figure 2.20. WIN 55,212-2 reduces both sEPSC and sIPSC frequency onto PND 9-10 Golgi cells. (A) Circuit diagram showing a whole-cell patch recording (Vh = -30mV) from Golgi cells. (B) Golgi cells were distinguished from granule cells visually based on their large size within the granule cell layer using DIC imaging (florescence shown for illustrative purposes), and by their relatively larger capacitance (Golgi area under curve: 413.2 pA*ms; GC area under curve: 75.0 pA*ms). (C) Inset representative traces and a quantitative graph showing a reduction in both Golgi cell sEPSC (inward) and sIPSC (outward) frequencies following 5µM WIN administration. ** indicates p<0.01

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FIGURE 2.21 A

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Figure 2.21. Circuit diagrams depicting CB1R expression (green) within the cerebellar cortex in both developing (PND 4-12) and mature (PND 29-36) rats. There are distinct CB1R expression profiles between developing and mature cerebellar cortex tissue. The dark grey neurons within the rectangle represent the main glutamatergic circuit through the cortex, with lighter shades of grey depicting inhibitory interneurons or climbing fibers synapsing directly onto Purkinje cells. Green axons represent where electrophysiological experiments have shown CB1Rs to be expressed. A) CB1R expression in the developing (PND 4-12) cerebellar cortex, with expression at the following synapses: Mossy Fibers->Granule cells; Golgi cells->Granule cells; Granule cells->Golgi cells; Golgi cells->Golgi cells; Stellate/Basket cells->fellow Stellate/Basket cells, and/or potentially onto Golgi cells. Climbing fibers are assumed to express CB1Rs based on previous literature. B) CB1R expression in the mature (PND 19-36) cerebellar cortex, with expression in the following synapses: Granule cells- >Purkinje cells; Golgi cells->Granule cells; Stellate/Basket cells->Purkinje cells. Climbing fiber and Stellate/Basket cell axonal CB1R expression onto Purkinje cells is assumed based on previous literature. We did not record from mature Golgi cells and Stellate/Basket cells, so their expression is currently unknown and depicted with question marks.

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TABLE 2.1

PND 6-10 PND 30-34

Wildtype sEPSCs Cont. 15.1 ± 1.5 pA Cont. 14.1 ± 1.1 pA WIN 13.5 ± 1.2 pA WIN 14.0 ± 1.4 pA P=0.24 P=0.67 sIPSCs Cont. 10.0 ± 1.6 pA Cont. 8.6 ± 1.1 pA WIN 8.6 ± 0.7 pA WIN 9.7 ± 1.7 pA P=0.24 P=0.24 CB1R Knockout sEPSCs Cont. 13.9 ± 1.4 pA Cont. 10.8 ± 0.7 pA WIN 12.6 ± 1.1 pA WIN 11.1 ± 0.6 pA P=0.12 P=0.46 sIPSCs Cont. 8.8 ± 0.9 pA Cont. 7.0 ± 0.9 pA WIN 8.2 ± 0.9 pA WIN 7.0 ± 0.9 pA P=0.33 P=0.98

Table 2.1. Mean sPSC amplitudes in granule cell recordings of wildtype and transgenic CB1R knockout mice. There were no significant changes in sPSC amplitudes following bath application of the CB1R agonist WIN 55,212-2 (5µM). Displayed p- values are results from paired t-tests between control and WIN averaged sPSC amplitudes.

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DISCUSSION

By performing the experiments included within this chapter, we were able to determine the extent of CB1R expression and its actions on synaptic transmission within the developing and mature cerebellar cortex. This was accomplished by utilizing both immunohistochemical and electrophysiological techniques, in which we were able to visually identify CB1R expression in conjunction with other florescent markers, followed by pharmacological verification of their location using patch-clamp electrophysiology. It was reassuring that both our immunohistochemical and electrophysiological data in mature cerebellar tissue agrees with previous findings: we observed dense CB1R expression within the molecular layer and on basket cell terminals onto Purkinje cells, and report functional CB1R expression on parallel fiber afferents to Purkinje cells. These results give us confidence that our novel findings are supported by our ability to replicate previous observations, and provide confidence that our lack of effect in developing Purkinje cells was not an artifact or faulty experimental approach. We also report immunohistochemical CB1R expression within all layers of the developing cerebellar cortex, with especially dense expression surrounding the molecular layer, Purkinje layer, and the granule cell layer. When examining CB1R expression within the granule cell layer more closely, we were able to identify colocalization with glutamatergic markers VGluT 1 and 2 in the absence of the nuclear marker Hoechst, which suggests cannabinoid expression within granule cell layer glomeruli. The glomeruli is the point of synaptic contact between granule cell dendrites with mossy fiber and

Golgi cell terminals, so these data were supported by electrophysiological recordings showing the CB1R agonist WIN 55,212-2 reduces vesicle release in both synapses. We also confirmed actions of CB1R activation while recording from Golgi cells and stellate/basket cells, but

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surprisingly not Purkinje cells. These findings give credence to a functional role CB1Rs provide in coordinating novel developmental synaptic transmission, which may prove important in a number of developmental processes.

Immunohistochemical observations and electrophysiological confirmation

The results that can be taken from CB1R immunohistochemical images in developing tissue are largely in agreement with the electrophysiological data gathered when applying the CB1R agonist

WIN 55,212 within cerebellar tissue, confirming the effects of CB1R activation in a variety of neurons in which immunohistological CB1R expression was found. The observed CB1R expression was confirmed within granule cell layer glomeruli based upon reduced glutamatergic and

GABAergic vesicle release (EPSCs and IPSCs, respectively) from mossy fiber and Golgi cell terminals following WIN application. Similarly, Golgi cells, stellate, and basket cells all responded to WIN application, which is outlined in circuit diagrams for both developing and mature tissue

(Fig. 2.21). Yet there appears to be a discrepancy between the high CB1R expression in the molecular layer and the apparent lack of CB1R response to WIN in either Purkinje sEPSCs/IPSCs or eEPSCs/IPSCs. This is also counter to the strong inhibition of glutamatergic and GABAergic synaptic transmission onto more mature Purkinje cells following application of a CB1R agonist.

Interestingly, sEPSCs (from granule cell parallel fibers) were reduced when recording from Golgi cells, suggesting some parallel fibers within the molecular layer must express functional CB1Rs.

This discrepancy in CB1R activation—the fact that WIN reduces sEPSCs only in parallel fiber terminals onto Golgi cells (Fig. 2.20C)—may explain why there appears to be heavy CB1R expression in the molecular layer despite the lack of effect WIN has on Purkinje cell sEPSC activity.

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Another possible explanation for this discrepancy could be CB1R expression on stellate and basket cell terminals onto fellow stellate/basket cells. Our electrophysiological data found that

WIN reduces GABAergic vesicle release in these synapses (Fig. 2.19D), and since these synaptic connections are also formed within the molecular layer it is possible that immunohistochemical

CB1R expression found within the molecular layer could be at these terminals. Further imaging studies may be required to determine whether CB1Rs colocalize with specific sites, such as determining whether fluorescently-filled stellate/basket/Golgi cells colocalize with molecular layer puncta in developing tissue. Previous work has identified CB1R colocalization with the vesicular GABA transporter VGAT in PND 14 mice within the molecular layer161. Applying a similar approach may prove useful in determining whether CB1R expression colocalizes primarily with parallel fibers or inhibitory interneuron fibers. Finally, the discrepancy in molecular layer CB1R expression may be explained by the presence of nonfunctional CB1Rs, or CB1Rs playing a role outside of canonical synaptic mediation of vesicle release. Confirming this possibility may require molecular techniques to determine whether CB1R activation triggers novel intracellular mechanisms.

There also appears to be CB1R expression on immature granule cell somas within the external granule layer. Considering these immature neurons have yet to establish synaptic connections, it would be interesting to determine whether this immunohistochemical labelling indicates CB1Rs on these neurons provide a non-synaptic function. For example, the CB1R agonist

WIN 55,212-2 has been shown to activate CB1Rs in developing neurons and lead to growth cone retraction through an intracellular Rho-GTPase mechanism148. Similar non-synaptic CB1R functions exist within the developing cerebellum as well. Granule cell precursors within the

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developing cerebellum express functional CB1Rs, and their activation promotes granule cell precursor proliferation in vitro and in vivo through the CB1R/AKT/glycogen synthase kinase-3β/β- catenin pathway145. These results reveal multiple mechanisms in which CB1R activation can influence neurodevelopment, providing many veins of study to pursue relating to non-synaptic

CB1R function in addition to what we have examined within this Chapter.

Functional CB1R Expression: Granule Cells

Several groups have previously observed PND 6-8 cultured cerebellar granule cells respond to

CB1R agonists Δ9-tetrahydrocannabinol (THC)174,175 and WIN 55,212-2173. Unfortunately, cultured cells undergo a variety of molecular adaptations, so are not necessarily reflective of responses in acute, intact tissue. This limits the translational applicability to intact cerebellar cortex synaptic circuitry. We report that CB1R agonist WIN 55,212-2 acts to reduce both glutamatergic and

GABAergic synaptic vesicle release onto granule cells in developing (PND 6-12) tissue (Fig. 2.6 &

2.7), providing the first known example of cerebellar granule cell CB1R synaptic modulation in slice. The presence of functional CB1Rs on mossy fiber terminals provides an example of how the endocannabinoid system is able to modulate synaptic transmission through the primary circuit of the cerebellar cortex, which has the potential to modulate the excitability of multiple interneurons in addition to Purkinje cells. What perhaps makes this developmental phenomena more interesting is the observation that WIN still reduces GABAergic neurotransmission onto granule cells in mature (PND 28-33) tissue, yet fails to reduce mature glutamatergic mossy fiber vesicle release (Fig. 2.11B). This finding suggests functional CB1Rs on mossy fiber terminals may be eliminated following maturation of cerebellar tissue. The transient developmental expression

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of CB1Rs at mossy fiber terminals may provide additional control on granule cell excitability and subsequent parallel fiber glutamate release. This may serve an especially important developmental role while the cerebellar cortex is undergoing rapid morphological and synaptic changes. Glutamate is known to increase immature granule cell migration rate40,46, so provided that granule cell parallel fibers are the source of this glutamate it is likely that fine tuning the level of granule cell excitation through cannabinoid activation aids to regulate granule cell migration.

Our data may also suggest CB1R activation exhibits varying downstream intracellular mechanistic roles depending on the synapse in which it is expressed. For example, CB1R agonist

WIN reduces mossy fiber->granule cell eEPSC amplitude and sEPSC/mEPSC frequency, but Golgi cell->granule cell eIPSCs do not decrease in amplitude despite sIPSC/mIPSC frequencies reducing following WIN application. This seemingly contradictory result is perplexing, yet there may be an explanation to explain this discrepancy. First, it may prove enlightening to examine the results of previous studies in Purkinje cells. For example, in PND 16 Purkinje cells WIN (5µM) produces a large reduction in parallel fiber-stimulated eEPSC amplitude but fails to alter mEPSC frequency, which the authors conclude is due to effects on voltage-gated calcium channels at the terminal163.

Our own observations find a lack of WIN effect on granule cell eIPSC amplitude but reduces mIPSC frequency, which may elude to CB1R-activated mechanisms downstream of voltage-gated calcium channel activity. In other words, it may be possible that CB1Rs on Golgi cell presynaptic terminals only influences downstream vesicle-release machinery following Ca2+ release, but has no effect on voltage-gated P/Q, N, or R-type Ca2+ channels, as is the case in parallel fibers106,107.

CB1R actions only on inhibiting vesicle release machinery, rather than a combination of inhibition

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on both vesicle release machinery and voltage-gated calcium channels, may lessen the effectiveness CB1Rs have on reducing vesicle release probability. This theory agrees with our own observations in the following ways: First, 5µM WIN reduces sEPSC frequency by 64% compared with a 43% reduction in sIPSC frequency, which would agree with data showing higher CB1R effectiveness at glutamatergic terminals. Second, locally stimulated eEPSC amplitude is reduced in 5µM WIN (Fig. 2.6C & D), whereas eIPSC amplitude is not (Fig. 2.7C & D), which may suggest

Golgi cell stimulation is above a threshold in which a reduction of vesicle release probability would be observed. Third, the CB1R inverse agonist SR (1µM) only partially blocks the reduction in sEPSC frequency following 5µM WIN application, but fully blocks sIPSC frequency (Fig. 2.10B

& C). This may suggest a lower proportion of CB1R activation is needed to reduce sEPSC frequency in mossy fiber terminals, whereas a high proportion of CB1Rs on Golgi cell terminals must be activated to achieve the same result. The difference may be that CB1R activation suppresses excitatory vesicle release more effectively than at inhibitory terminals161.

The discrepancy between the contrasting blocking efficacies of the CB1R inverse agonist

SR141716A (SR; 1µM) in granule cell glutamatergic mossy fiber and GABAergic Golgi cell synapses may also suggest CB1Rs at these synapses could differ in their intracellular mechanisms. The 1µM

SR concentration fully blocked a reduction in sIPSC (Golgi) frequency following 5µM WIN application, whereas sEPSCs (mossy fibers) required a greater concentration (2µM SR) to achieve a complete block. This may imply differing CB1R-mediated intracellular mechanisms controlling vesicle release between Golgi and mossy fiber terminals. Since Golgi cell eIPSC amplitude did not reduce following WIN application, which implies CB1Rs may mediate vesicle release downstream of Ca2+ release at Golgi terminals, it is possible the CB1R inverse agonist SR may provide a

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complete block because downstream Ca2+ CB1R mechanisms play a smaller role in inhibiting vesicle release. In other words, there may be a lower threshold of CB1R activation needed to inhibit VG channels, taking a higher [SR] to block WIN-induced inhibition—VG channels are more easily inhibited by CB1R activation, making the effect of WIN harder to block in mossy fibers, which would explain the incomplete block with 1µM SR. When considering the dose response following WIN application in the presence of SR141716A, a higher concentration of SR141716A

(e.g. 8µM vs 1µM) had a higher percent block in both mossy fiber and Golgi cell terminals onto granule cells. To add to the complexity, 1µM SR was able to completely block a WIN-induced reduction in eEPSC amplitude, while it was unable to block a reduction in sEPSC frequency. It is possible that mossy fiber stimulation activated mossy fiber collaterals lacking CB1Rs, which were able to override any WIN-mediated reduction in eEPSC amplitude. However, if this were a common phenomenon it would be unlikely we see such pronounced reductions in eEPSC amplitude when simply bath applying WIN. This may require additional experiments to completely explain.

It is also important to address the inability of synaptic activity to recover from blunted signaling in response to WIN 55,212-2 following a washout condition. For the sake of clarity we excluded the Wash condition from data figures, but it clearly needs to be addressed. As mentioned earlier, cannabinoids are notoriously “sticky,” which likely explains the inability to for

WIN to properly wash following experimental manipulation. We attempted to ensure the high

CB1R affinity and lipophilicity of WIN 55,212-2 was the issue for the lack of wash by attempting to displace WIN with the CB1R inverse agonist SR141716A in its presence. If effective, SR should return synaptic activity to baseline conditions, which is essentially a different way to observe the

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ability of SR to block the effects of WIN, but easier to conceptualize. Bath application of 1µM

SR141716A following a WIN-induced reduction in synaptic transmission caused sEPSC and sIPSC frequencies to recover, completely or partially, to baseline levels (Fig. 2.13). This is interesting because there results are essentially the opposite of those following application of 5µM WIN in the presence of 1µM SR141716A. For example, WIN was able to partially reduce synaptic sEPSC frequency in the presence of 1µM SR (Fig. 2.10B), whereas 1µM SR was able to partially restore a WIN-induced reduction in sEPSC frequency (Fig. 2.13A), showing that a 1µM dose of SR is not as effective at either blocking actions of WIN or displacing WIN from CB1Rs. It is also interesting that higher doses of SR141716A, although not statistically significant, show a trend of increased sEPSC and sIPSC frequency compared to lower doses (8µM vs 1µM) (Fig. 2.10B & C). Essentially, in addition to blocking WIN-induced synaptic suppression SR may also be increasing the baseline response. As noted previously, it is difficult to theorize if a classical antagonist, rather than an inverse agonist, would have the same effect. As an inverse agonist, SR141716A may have the potential to further inhibit the CB1R past baseline levels rather than simply displacing WIN from the ligand-binding pocket of CB1Rs. Although these experiments are useful for confirming the

WIN-induced reduction in synaptic transmission is a real effect, repeating these experiments using a classical antagonist would be more enlightening in determining the underlying cause driving the inability of synaptic transmission to recover from the effects of WIN administration.

Functional CB1R Expression: Purkinje Cells

We also investigated whether the CB1R agonist WIN could alter Purkinje cell synaptic transmission in developing tissue. In addition to developmental differences found at granule cell

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synapses, WIN’s inability to reduce the probability of vesicle release in either excitatory parallel fibers or inhibitory basket and stellate cells when recording from Purkinje cells was equally surprising. This was especially surprising when considering the heavy modulatory role CB1Rs play at these synapses in mature tissue, coupled with what appears to be heavy molecular layer CB1R expression in our immunohistochemistry studies. It is difficult to speculate why the cerebellar cortex may have evolved to lack functional developmental CB1R expression at these synapses, but provides an additional example of endocannabinoid adaptability to suit developmental needs.

There are instances of endocannabinoids exhibiting tonic activation of CB1Rs in a number of cell types. For example, CB1Rs regulate GABA release in vasopressin neurons within the paraventricular nucleus of the hypothalamus (PVN), where blocking CB1R activity ultimately increases sIPSC frequency227. Also, the CB1R antagonist AM251 and inverse agonist SR141716A increase mIPSC frequency in PND 9-14 mouse Purkinje cells165, and AM251 increases PND 14-21 rat stellate/basket cell firing rate beyond baseline frequencies166. Therefore it is possible that blocking the actions of CB1Rs could reveal intrinsic CB1R activation by endocannabinoids. Our results show bath application of 2µM SR141716A in PND 8-10 Purkinje cells slightly increases sEPSC frequency (Fig. 2.18). It is possible there is tonic CB1R regulation of glutamate release at the parallel fiber->Purkinje cell synapse. However, the fact that CB1R agonist WIN failed to reduce synaptic transmission onto Purkinje cells makes an increase in sEPSC frequency following

SR141716A application surprising. A possible explanation may be that CB1Rs are maximally activated during baseline conditions, preventing WIN from having additional effects. A second, potentially more plausible possibility lies in the fact that SR141716A is an inverse agonist, which

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would allow it to inhibit constitutively active CB1Rs below their baseline state, making it more favorable for CB1Rs (and their associated G-proteins) to be in their inactive configuration.

Repeating experiments using a classic antagonist (e.g. AM 251) may aid us in determining whether simply blocking CB1Rs increased sEPSC frequency, yet previous recordings from Purkinje cells found AM251 increased the firing frequency of PND 14-21 rat interneurons166, which was not observed in interneurons using SR141716A in PND 18 rats167. Developmental age may be a contributing factor in response to a CB1R antagonist, as is network effects (e.g. a CB1R antagonist may block a CB1R-mediated decrease in glutamatergic transmission onto interneurons, increasing its excitability). Our stellate/basket cell data show CB1R agonist WIN reduces sIPSC frequency and has no effect on sEPSC frequency, so those results would suggest a decrease in excitability, if anything. Characterizing functional CB1R expression in stellate/basket cells within the same developmental age as the aforementioned studies may aid in interpreting this data.

Despite the well characterized CB1R expression at mature Purkinje cell synapses, the ultimate purpose of transitory cerebellar CB1R expression in vivo lacks clarity. Contrary to what one might expect, CB1R knockout mice appear to exhibit few major deficiency phenotypes228,229.

Despite CB1R KO-induced deficits in neurodevelopmental processes such as neurogenesis147 and misrouting of neuronal migration144, gross behavioral phenotypic differences may be masked.

Yet eyeblink conditioning has still been found to be impaired in CB1 knockout mice230, which may provide evidence to support a disruption in cerebellar development. This is an example of how a lack of CB1R signaling may impact cerebellar development, but overactivation of CB1Rs can also prove detrimental. For example, exogenous cannabinoid treatment in pregnant rats leads to motor impairment in PND 22 offspring68 and altered migration of glutamatergic and GABAergic

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cerebral neurons193. Considering the adaptability of the endocannabinoid system, it is worthy to consider potential compensatory effects of knocking out the CB1 gene. Examining the effects of exogenous cannabinoids in a third trimester equivalent rodent model may reveal the brain is unable to quickly adapt to an acute form of altered synaptic signaling. This may provide reason to believe that temporally specific CB1R dysregulation may be more damaging to neurodevelopment, as the brain has not had adequate opportunity to adapt to a change in signaling.

Conclusions

Our investigation of CB1R expression and synaptic modulation has disseminated only a small component of how the endocannabinoid system may function within the developing cerebellum.

Overall, our data provides novel insights into CB1R distribution and actions within the developing cerebellar cortex: the expression of CB1Rs throughout the developing cerebellar cortex is quite ubiquitous, found within all layers, but both spatially and temporally specific to certain neurons at a variety of synapses. We have found functional CB1R expression at the Golgi->granule synapse, the Mossy fiber->granule cell synapse, and synapses of inhibitory interneurons, all maintaining a role as presynaptic vesicle-release modulators where they reduce the probability of vesicle release and synaptic strength. These data are the first instance comparing synaptic

CB1R function across a developmental timespan within slice tissue, which provides a critical understanding of how cannabinoids influence synaptic transmission within a defined neuronal network. However, there may be CB1R expression beyond the synapses we investigated according to the pervasiveness of CB1R expression in our immunohistochemistry studies For

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instance, CB1R expression may be located somatically and exhibit non-synaptic functions, such as increasing granule cell precursor proliferation within the external granule layer145. Yet our findings lay the groundwork for future investigation of molecular impacts CB1Rs have on developmental processes seen in other brain regions, such as neuronal differentiation, axonal pathfinding, and synaptogenesis148,193,231–233, in future cerebellar work.

Overall, these data provide evidence of transient CB1R expression within key synapses as neurodevelopment occurs, while exemplifying the strong synaptic control CB1Rs have over glutamatergic and GABAergic signaling, which highlight the importance of the endocannabinoid system during the most active period of cerebellar development. In the following chapter, we attempt to determine the how CB1R activation on granule cell afferents may influence polysynaptic signaling and glutamatergic throughput onto Purkinje cells and migrating granule cells. This will provide insight into how the modulatory effect of CB1R activation on synaptic signaling can alter signaling molecules known to influence cerebellar developmental processes.

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CHAPTER 3

CB1R mechanism and effect on cerebellar polysynaptic network activity. Jesse L. Barnes, Hiroko Shiina, and David J. Rossi

ABSTRACT

There are an intricate series of developmental processes that occur in order for neurons within the cerebellar cortex to properly proliferate, differentiate, migrate, and form appropriate synaptic connections post-migration. Granule cells rely on a number of chemical triggers including glutamate to fine tune correct migratory speed and position within the granule cell layer (GCL). In the previous chapter we demonstrated that CB1R activation on mossy fiber and

Golgi cell terminals reduces synaptic vesicle release onto granule cells, which likely modulates granule cell excitability and subsequent glutamate release from granule cell parallel fibers. In this chapter we determine that our previous finding, that reduction in mossy fiber-stimulated glutamate release onto granule cells using CB1R agonist WIN 55,212-2 (WIN), results in attenuated glutamate release onto both Purkinje cells and migrating granule cells, which indicates CB1R activation maintains a polysynaptic influence on downstream targets. These findings have implications for the proper execution of sensitive developmental processes reliant on appropriate glutamatergic cues, cues which ensure proper dendritic arborization, synaptogenesis, and cell migration and whose disturbance may result in long-term behavioral implications.

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INTRODUCTION

The cerebellar cortex is complex yet highly organized, and comprised of billions of neurons with two main glutamatergic inputs: the mossy fiber input, which innervates granule cells, and climbing fibers, which directly synapse onto Purkinje cells. Glutamatergic granule cells synapse onto a variety of GABAergic interneurons in addition to Purkinje cells, thus modulatory control over their excitability has widespread influence over signaling within the cerebellar cortex. The endocannabinoid system may have such an influence. As we have seen in Chapter 2, the CB1Rs are heavily influential on synaptic signaling in a multitude of cerebellar synapses, with functional

CB1Rs found on synaptic terminals onto all major neuronal cell types within the cerebellar cortex with the exception of Purkinje cells. We know that activating CB1Rs on terminals presynaptic of recording cells reduces vesicle release probability, but it is currently unknown whether that reduction in vesicle release can influence the excitability of cells and subsequent downstream vesicle release. In the neurons of developing tissue with presynaptic CB1R modulation of both glutamatergic and GABAergic afferents, such as granule cells, it may be considered difficult to predict the summed outcome of granule cell excitability and subsequent glutamate release on downstream targets. We can infer that overall excitability would be dampened considering a reduction in glutamate release, and if GABA is excitatory in early development, reduced GABA release would dampen excitation of granule cells as well. However, as development progresses and GABAARs no longer produce depolarizing currents (around PND 8), there is presumably a shift in granule cell excitability following CB1R activation.

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There are a number of ways the endocannabinoid system has been implicated in neurodevelopment. The endocannabinoid 2-AG has been found to facilitate the migration of pyramidal cells and guide axonal projections234. Overexpressing sn-1-diacylclycerol lipase α

(DAGLα), the enzyme that synthesizes the endocannabinoid 2-AG, can repulse cholinergic growth cones in vitro151. This suggests intracellular endocannabinoid signaling may occur to guide cholinergic neuronal projections, and may ultimately affect synapse positioning. Additionally, inhibiting normal CB1R function in chick embryos leads to problems in axon pathfinding235.

Exposing embryonic CB1R-transfected kidney cells to exogenous cannabinoids can promote the induction of migration149. Endocannabinoid signaling may also indirectly alter guidance cues responsible for developmental processes such as synaptogenesis. For example, NMDA receptor- based synaptic contacts play an integral role in the development and maintenance of synapse formation and elimination in the hippocampus236. Purkinje cells are also affected by changes in glutamate levels, as Purkinje dendritic arborization in mice is abnormal when lacking glutamate signaling60,61. Purkinje cell spine formation is nearly halved in mutant mice lacking the GluRδ2 subunit, and many of the spines present had no terminal attachment62. This provides further justification in studying how the CB1R-induced reduction in upstream glutamate release at mossy fiber terminals can dampen glutamate levels, which may act to alter multiple aspects of normal development. However, we mainly focus on the role of synaptic output on known guidance cues for granule cell migration.

Glutamate has also been implicated in developmental processes such as migration, as ambient glutamate levels are known to manipulate the speed of immature granule cell migration40,45. Granule cell parallel fibers release glutamate in close proximity to granule cells

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migrating trough the molecular layer, thus CB1R activation on granule cell afferents may reduce granule cell excitability and subsequently diminish parallel fiber glutamate release. Granule cell migration rates can also be regulated by the activation of voltage-gated Ca2+ channels (VGCCs), which NMDA activation may also alter and indirectly increase migration rates. Granule cells begin to express primary N-type Ca2+ channels as they reach the bottom of the external granule layer.

Using an N-type specific Ca2+ channel blocker slows molecular layer migration rate, whereas blocker for L- and T-type channels have no effect on migration rate46. Reducing Ca2+ influx by reducing extracellular Ca2+ or blocking NMDA receptors decreases the size of intracellular Ca2+ fluxuations, which correlates with reduced neuronal migration47. Additionally, overexpressing the NR2B NMDA receptor subunit in migrating granule cells increases granule cell migratory speed237, which may partially explain the aforementioned results when considering NMDA receptors activate VGCCs. Granule cell precursors differentiate within the external granule layer and eventually migrate to their final post-migratory destination within the granule cell layer during the third trimester equivalent developmental period in rodents (PND 4-12). We have demonstrated granule cells receive modified synaptic signaling following CB1R activation during this period, so extrapolating these data suggest a CB1R-mediated reduction in glutamate release onto migrating granule cells may slow granule cell migration rate, which may impact their ultimate positioning.

In the previous chapter, we found that the CB1R agonist WIN 55,21-2 reduces vesicle release onto granule cells, but has no effect on parallel fiber vesicle release onto Purkinje cells.

Here we attempt two different approaches to determine whether upstream CB1R activation at granule cell presynaptic terminals (as explored in Chapter 2) is able to modulate polysynaptic

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vesicle release from glutamatergic parallel fibers onto both Purkinje cells and migrating granule cells. A change in glutamate release onto these targets may have implications on how the cerebellar cortex forms at various developmental steps, such as neuronal migration and synaptogenesis, processes thought to contribute to the pathogenesis of neurodevelopmental disorders such as autism spectrum disorder238, epilepsy239, and schizophrenia240.

METHODS

Animals. All animals were bred and housed within a Washington State University (WSU) vivarium.

All procedures conformed to regulations approved by the Washington State University

Institutional Animal Care and Use Committee (IACUC), and conformed to all guidelines for ethical protocols and care of experimental animals established by the National Institutes of Health,

Maryland, USA. Sprague Dawley rats (Simonsen) were used for all experiments.

Acute preparation of brain slices. Rodent ages were either between postnatal day (PND) 4-12 for third-trimester equivalent rats. Pups (PND 4-12) were housed with their dams. All animals were kept on a standard light/dark cycle with lights on at 07:00. Cerebellar slices of randomly selected male or female rodents were prepared each day of experimentation. Rodents were anesthetized with isofluorane and decapitated, with brain extraction occurring in 1°C aCSF containing 124 mM NaCl, 26 mM NaHCO3, 1 mM NaH2PO4, 2.5 mM KCl, 2.5 mM CaCl2, 2 mM

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cerebellar vermis were taken using a Leica VT1200S vibratome in a slicing chamber filled with bubbling 1°C aCSF. Slices were then incubated in aCSF containing 1 mM kynurenic acid at physiological temperature (34-35°C) for 1 hour before electrophysiological recordings were conducted.

Slice Electrophysiology. Slices were secured with a platinum harp in a submersion chamber mounted on an Olympus BX51WI microscope, and visualized with a 60X (0.90 N.A.) water- immersion objective. Slices were perfused at a rate of 5-7 ml/min with artificial cerebrospinal fluid (aCSF), maintained at a temperature between 32-36° C, and bubbled with a 95% O2/5% CO2 gas. Purkinje cells and migrating granule cells were visually identified and voltage-clamped (Vh =

-30mV) with patch electrodes made from borosilicate glass capillary pipettes. Pipettes contained:

130 mM CsGluconate, 4 mM NaCl, 0.5 mM CaCl2, 10 mM HEPES, 5 mM EGTA, 4 mM MgATP, 0.5 mM Na2GTP, and 5 mM QX-314. Internal solution was pH-adjusted to 7.2-7.3 with CsOH.

Electrode resistance was 4-8 MΩ for granule cells and 1-4 MΩ for Purkinje cells. Cells were excluded if access resistance changed by >20% throughout the duration of an experiment. Given

- the extra- and intracellular [Cl ], ECl was ~ -60mV, so voltage-clamping at -30mV resulted in IPSCs being outward and EPSCs being inward. All recordings were filtered at 10 kHz and acquired at

20kHz. A glass capillary stimulating electrode was used to evoke action potentials in mossy fibers, with a stimulation range of 25µA-1.5mA, determined by an easily detectible and consistent amplitude evoked synaptic response. Drugs were dissolved in aCSF and constantly bubbled with

95% O2/5% CO2 gas before being administered. All experimental conditions were conducted for

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5 minutes each, with the baseline condition being conducted for at least 5 minutes or until a stable baseline was reached.

Migrating granule cell single channel electrophysiology. Mig-GCs in the molecular layer were selected visually based on their small size and tear-drop shape. Highly electrically-compact characteristics of mig-GCs (i.e. small somata, no dendrites and few channels) enabled single- channel voltage-clamp (Vh=-60mV) recordings in whole-cell configuration. To enable recording of single NMDAR channels at -60mV (required to get large enough single-channel currents to resolve in whole-cell mode), recordings were done in Mg2+-free aCSF, to relieve voltage- dependent Mg2+ block at such hyperpolarized potentials. Otherwise all solutions were the same as used for other recordings described above. To determine if afferent activity affected mig-GC

NMDA channel activity, a glass capillary stimulating electrode was placed in the white matter

(location of the mossy fibers), and stimulated (paired pulse, 20 ms inter-pulse interval) every 20 s at intensities ranging from 30 to 500 µA. Whole-cell voltage-clamp recordings were acquired and filtered as described above, and signals were further filtered at 1 kHz for visualization and analysis of single channel activity.

Analysis of evoked EPSC currents. Electrically evoked (1 stim/20 sec; 10 stim/2 min) synaptic currents were quantified using pClamp 10.4 (Clampfit; Axon Instruments, Foster City, CA). The final ten evoked responses in every condition were averaged, and the mean peak amplitude was calculated. Because the CB1R agonist WIN 55,212-2 fails to wash out within a reasonable amount of time, the wash condition is excluded in the following results unless noted otherwise.

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Mig-GC NMDAR-mediated single channel activity analysis. The measurements of NMDAR- mediated single channel activity was previously described 45. Briefly, NMDAR-mediated single channel activity was reported as a product of the number of channels and open probability. This was measured experimentally as the total change in current over 3s following stimulation. Mean single channel amplitude was determined by Gaussian fit of 100 manually selected openings during the baseline period.

Evoked NMDAR single channel current analysis. To determine the total current induced by mossy fiber stimulation, first, a 1s baseline region with no events was selected within a 5s period immediately before the electrical stimulation. The holding current during this baseline quiescent period was subtracted from the entire trace. Then, the area of the trace 3s immediately following stimulation was calculated, which provided the total evoked current (ms*pA) induced by stimulation. This response is measured as the current (pA) over this 3s period, divided by single channel amplitude, and reported as the product of the number of channels (n) and the open probability (p).

Statistics. Data are expressed as the means ± the standard deviation of the means (M ± SEM) for each condition. Within-cell evoked PSC amplitudes and total NMDA evoked current before and after drug treatments were compared using paired students t-tests. Significance was p<0.05 unless otherwise noted.

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Reagents. Kynurenic acid (1mM; Abcam; ab120064) was added to aCSF solution during brain extraction. Isolation of eEPSC currents was accomplished using 10 µM GABAzine (Abcam; ab120042). To block NMDA channel activity, 50µM AP5 was used (Abcam; ab120003). The CB1 agonist (+)-WIN 55,212-2 was purchased from Cayman Chemical (Cat. No. 10009023).

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RESULTS

Polysynaptic Purkinje cell signaling

MF-evoked EPSCs

We examined whether a CB1R-mediated reduction in vesicle release at the mossy fiber to granule cell synapse affects downstream Purkinje cell excitation. This was achieved by recording from whole-cell voltage-clamped PND 4-10 rat Purkinje cells (Vh=-30mV) while electrically stimulating glutamatergic mossy fibers (Fig. 3.1A). Mossy fiber stimulation was able to elicit polysynaptic eEPSCs in Purkinje cells (baseline amplitude (pA) = 280.2 ± 69.9) (Fig. 3.1B), and bath application of CB1R agonist WIN 55,212-2 (5µM) decreased polysynaptic eEPSC amplitude (% decrease = 45.5

± 6.5, p<0.01, n = 10) (Fig. 3.1C). These results demonstrate that CB1R activation on granule cell afferents are able to collectively reduce granule cell activation, and ultimately glutamatergic release, onto Purkinje cells. In order to isolate the glutamatergic mossy fiber -> granule cell circuit without the influence of GABAergic interneurons, the same experiments were done in the presence of the GABAAR antagonist Gabazine. The addition of Gabazine (10µM) did not reduce baseline polysynaptic eEPSC amplitude (mean baseline amplitude (pA) = 383.25 ± 98.15), but as expected, the addition of WIN (5µM) in the presence of Gabazine (10µM) reduced eEPSC amplitude (% decrease = 42.2 ± 6.7, p<0.05, n = 11) (Fig. 3.1D). This experiment resolves the question over whether GABAergic Golgi afferents onto granule cells influence their excitability, finding granule cell excitability fails to change in the absence of Golgi cell actions.

Polysynaptic migrating granule cell signaling

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To determine whether CB1R actions at the mossy fiber to granule cell synapse also mediate glutamate release onto migrating granule cells, my colleague Hiroko Shiina whole-cell patch- clamped migrating granule cells (mGCs) (Vh=-60mV) located within the molecular layer of PND 5-

8 rats while electrically stimulating mossy fiber afferents. The mGCs were distinguished by their teardrop shape and extending neurites (Fig. 3.2B for DIC image overlay). Recordings revealed mGCs exhibited spontaneous single channel NMDAR channel activity (mean single channel current amplitude = 5.1 ± 0.1 pA) (Fig. 3.2Ca), while electrical stimulation of mossy fibers evoked a large transient increase in single channel NMDAR activity, seen by a large inward current (Fig.

3.2C & D). Application of NMDAR antagonist AP-5 (50µM) blocked spontaneous and evoked single channel NMDAR activity, confirming this activity was NMDA receptor specific (Fig. 3.2C &

Cb). Bath application of the CB1R agonist WIN 55,212-2 reduced glutamate release onto migrating granule cells, seen as a reduction in single channel NMDAR activity (% reduction = 57.7

± 9.3, p<0.01, n=13) (Fig. 3.2D). These findings support the hypothesis that activation of CB1Rs on granule cell mossy fiber afferents dampens mossy fiber excitability of granule cells. This reduces mossy fiber-evoked granule cell action potential firing, thereby reducing parallel fiber glutamate release into the molecular layer.

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FIGURE 3.1

A B

C D

Figure 3.1. CB1R agonist WIN polysnaptically reduces mossy fiber-evoked glutamate release onto Purkinje cells in the developing (PND 5-8) cerebellar cortex. (A) Circuit diagram depicting a voltage-clamped Purkinje cell (Vh = -30mV) while electrically stimulating mossy fibers (MFs). The polysynaptic signal must propagate through the MF to granule cell synapse before reaching the parallel fiber to Purkinje cell synapse. (B) Overlayed representative traces showing 5µM WIN application reduces MF-evoked polysynaptic eEPSC amplitude. (C) Quantification of reduced Purkinje polysynaptic mean eEPSC amplitudes following 5µM WIN administration. (D) GABAAR antagonist Gabazine (10µM) alone fails to reduce baseline MF- evoked polysynaptic eEPSC amplitude, indicating GABAergic minimal GABAergic influence on Purkinje cell glutamate release.

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FIGURE 3.2 A B

C D

E F G

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Figure 3.2. CB1R agonist WIN reduces Mossy Fiber stimulated increase in NMDA receptor channel activity in migrating granule cells of PND 5-8 rats. (A) Circuit diagram depicting a voltage-clamped (Vh=-60mV) migrating granule cell (mGC) while electrically stimulating mossy fibers (MFs). MF-evoked granule cell action potential firing triggers glutamate release onto mGCs. (B) DIC image with cartoon overlay depicting mGC recording while stimulating MFs. Note the relatively large size of the external granule layer compared to the molecular layer in this slice of PND 5 rat. (C) Representative mGC trace showing spontaneous and mossy fiber-stimulated single channel NMDAR openings, which are blocked with the NMDAR channel blocker AP5 (50µM). (D) The average amplitudes (pA) of spontaneous single channel NMDAR openings. (E) MF-evoked NMDAR channel activity is blocked following application of NMDAR channel blocker 50µM AP5. (F) Overlayed representative traces showing decreased MF-evoked single channel NMDAR activity following 5µM WIN application. (G) Quantification of 5µM WIN-induced decrease in MF-evoked single channel NMDAR activity.

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DISCUSSION

In this Chapter, we initially show that activating CB1Rs on mossy fiber terminals reduces excitation of postmigratory granule cells, which in turn reduces glutamate release onto Purkinje cells. These findings provide novel insights into how cannabinoid receptor function during development, and also provides a more fundamental observation as the first known example showing the mossy fiber -> granule cell -> Purkinje cell circuit is polysynaptically connected during the most active period of cerebellar cortical development. Polysynaptic transmission through the cerebellar cortex has been previously unexplored, and our findings reveal robust parallel fiber glutamate release onto Purkinje cells.

Our results that show a CB1R-mediated polysynaptic glutamate reduction onto Purkinje cells provided several insights and potential implications. First, which was already alluded to, was it shows that mossy fiber stimulation is able to polysynaptically increase induce parallel fiber glutamate release onto Purkinje cells. The establishment of network activity within the cerebellar cortex was previously unknown, which this result verifies. Second, our data showing GABAAR antagonist Gabazine has no effect on baseline levels of polysynaptic eEPSC amplitudes implies

Golgi cells may have minimal influence over granule cell excitability. Since these recordings were performed in PND 5-8 rats, such results are enlightening in understanding whether GABAARs, if depolarizing, may influence MF-evoked polysynaptic Purkinje cell EPSC amplitude. However, the timing of Golgi cell-mediated GABA release onto granule cells would likely show a delayed onset since Golgi cell GABA release would be predicated on their excitation by mossy fiber-evoked parallel fiber activation. Third, a CB1R-mediated reduction in glutamate release onto Purkinje cells may potentially alter Purkinje cells development. Glutamate may be an important

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developmental signaling molecule for the Purkinje cell dendritic proliferation and synaptogenesis. For example, mice lacking a majority of granule cells display abnormal Purkinje cell dendritic arborization60, Purkinje cell NMDAR activation promotes dendritic differentiation through intracellular Ca2+ increases61, and Purkinje cell spine formation is stunted in GluRδ2- deficient mice62. Glutamate clearly plays at least a partial role in guiding Purkinje cell dendrites, which are concurrently developing with granule cells and during the time we observe these polysynaptic network effects. Forth, and perhaps most importantly, our initial experiments showing a reduction in glutamate release from parallel fibers onto Purkinje cells suggested there may also be a reduction in glutamate levels onto migrating granule cells.

Our observation that stimulating mossy fibers released parallel fiber glutamate onto migrating granule cell NMDARs was exciting, since their source of glutamate had been previously unknown. Since factors such as NMDA antagonists slow granule cell migration rate, and both the absence of magnesium and elevated levels of calcium increase migration rate40, a reduction in the amount of glutamate available to bind to migrating granule cell NMDA receptors may consequentially delay neuronal migration. Our second experiment revealed that CB1R activation reduces mossy fiber-evoked glutamate release to dampen mossy fiber excitation, thus reducing parallel fiber glutamate release onto migrating granule cells. We may make the assumption that altered levels of ambient glutamate levels following CB1R activation potentially impact granule cell migration rate, yet this experiment is the first of many needed before reaching a definitive conclusion. The first step is to establish that CB1Rs can, in fact, mediate glutamate release onto migrating granule cells before measuring migration rate directly.

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In addition to measuring mossy fiber-evoked single channel NMDAR activity in migrating granule cells, determining whether CB1R activation alters basal spontaneous single channel

NMDAR activity may reveal whether migration rate can change. The presence of the CB1R agonist

WIN may change overall ambient levels of glutamate release onto migrating granule cells regardless of the presence of evoked release, which may be evident in changes to the frequency of single channel openings. Multiple studies have shown migration rate is influenced by NMDAR activity40,45,49,241, which decreases following addition of NMDAR antagonists. A CB1R-mediated reduction in endogenous glutamate, coupled with reduced spontaneous single channel NMDAR activity, may emulate results from previous studies and reduce granule cell migration rate as well.

There is also the possibility that we are making the assumption that reduced glutamate levels onto migrating granule cells following CB1R activation is due to reduced granule cell excitability. However, there is still a possibility that CB1Rs are expressed on glutamate-releasing parallel fiber “terminals,” which may locally reduce glutamate release onto migrating granule cells. So far, none of our data can sufficiently conclude that the reduction in NMDA channel opening is due to CB1R activation in mossy fibers rather parallel fiber terminals themselves. It is easy to assume parallel fibers lack CB1R expression since parallel fiber->Purkinje terminals seem to lack expression. Our results showing CB1R activation on parallel fiber synapses reduces glutamate release onto Golgi cells clearly shows parallel fibers do appear to express active CB1Rs on some terminals. To determine whether there are also CB1Rs on the presynaptic terminal, we may need to locally stimulate parallel fibers while recording from migrating granule cells.

Granule cell migration rates can also change by manipulating levels of Ca2+ & cAMP and insulin-like growth factor 1 (IGF1) signaling242 within granule cells, which has been previously

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established using extrinsic pharmacological manipulation. Our data provides what we assume to be the first ex vivo account of polysynaptic glutamate release, and upstream manipulation via

CB1R activation, in which granule cell migration rate could be altered. Clearly, the need exists to examine these possibilities further since it is likely that glutamate is only one of many migrational mediators. One technique to examine these possibilities includes using time-lapsed ex vivo confocal imaging to track the rate of migrating neurons, as it would provide a more informative, collective means to characterize migration rates, rather than inferring changes in migration simply by measuring glutamate levels. This exists as a viable technique to determine whether we can bridge the association between CB1R-induced reduction in glutamate release, and the established observation that lower glutamate levels can slow granule cell migration.

The experimental manipulations carried out within this chapter enable us to understand how CB1R activation has the potential to change synaptic signaling, but this is less informative when attempting to compare our results to a functional in vivo endogenous system. An important component of characterizing the endocannabinoid system within the cerebellar cortex, and to attempt to understand the function of CB1Rs within a synapse, is to activate CB1Rs endogenously through the production of endocannabinoids. The primary endocannabinoids anandamide and

2-arachidonylglycerol (2-AG) are typically produced as needed within the postsynaptic density in response to membrane depolarization. A number of studies have been successful in provoking cerebellar endocannabinoid production by strong depolarizing voltage trains in Purkinje cells and observing whether this can reduce vesicle release into the synapse. For example, endogenous cannabinoids were found to induce reduced vesicle release in inhibitory stellate and basket cells133,134 in addition to a reduction in excitatory parallel fiber and climbing fiber activity136,137.

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Of course, these studies were performed in older cerebellar tissue at an age when parallel fibers and inhibitory interneurons presynaptic to Purkinje cells respond to CB1R agonists. In future experiments, there may be value in performing similar experiments in synapses we have determined to contain active CB1Rs, and determining the types of endocannabinoids produced at each synapse would greatly expand on our current findings.

Conclusions

The data developed within this chapter reveal an active and established primary glutamatergic circuit within the developing cerebellar cortex. In rats as young as PND 5, stable synaptic connections allow mossy fiber activation to enhance the excitability of post-migratory granule cells, which are then able to subsequently release glutamate onto distant downstream Purkinje and migrating granule cell terminals. These findings are novel by demonstrating mossy fiber actions are able to polysynaptically trigger glutamate release from parallel fibers. Also, our observation that mossy fiber stimulation polysynaptically increases single channel NMDAR activity on migrating granule cells, presumably from parallel fiber glutamate release, provides an explanation regarding their source of glutamate, which was previously unknown. Additionally, the results we have gathered within this chapter regarding cannabinoid function—showing upstream CB1R activation on mossy fiber terminals reduces downstream polysynaptic glutamate release onto multiple parallel fiber targets—provides evidence to support a functional role for the endocannabinoid system in polysynaptically modulating downstream glutamatergic synaptic transmission. Considering developmental synaptic processes appear to be influenced by

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glutamatergic signaling, CB1R activation may modulate these processes, and exogenous cannabinoid exposure may disrupt normal developmental functioning.

The presence of the CB1R agonist WIN may change overall ambient levels of glutamate release onto migrating granule cells regardless of the presence of evoked release, which may be evident in changes to the frequency of single channel NMDAR openings. Our electrophysiological findings, although informative from a mechanistic context, lack perspective regarding potential phenotypic consequences on granule cell migration, lobe formation, and behavioral outputs. As previously stated, determining CB1R-induced attenuation of glutamate release slows granule cell migratory rate following CB1R activation would serve to provide a clear implication of exogenous cannabinoid exposure during fetal development on a variety of behavioral phenotypes, as we discuss in chapter 4.

Of course, the current trend toward national legalization of cannabis within the United

States leads us to question how exogenous cannabinoids could potentially perturb cerebellar signaling during a developmentally sensitive time period, as there is a current lack of regulations and/or guidelines concerning cannabis consumption during pregnancy and adolescence. One of the first steps to understanding the effects of cannabis exposure during development is to characterize how it affects cell signaling within a specific brain region, and the behavioral output caused by this effect. In the following chapter, we aim to characterize phenotypic abnormalities associated with exogenous cannabinoid exposure during the cerebellar growth spurt as a measure of perturbation following cannabinoid consumption in pregnant mothers.

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CHAPTER 4

Effects of exogenous cannabinoid exposure in perinatal rats (long-term impacts). Jesse L. Barnes and David J. Rossi

ABSTRACT

Previously we have reported extensive expression of functional CB1R expression in multiple synapses within the developing cerebellar cortex, some of which appear to be transiently expressed. We have also found that CB1R activation at the mossy fiber to granule cell synapse dampens mossy fiber excitation of granule cells, reducing granule cell parallel fiber glutamate release onto developing Purkinje cells and migrating granule cells. Previous literature has found altered glutamate levels influence granule cell migration rate40,49 , neurite outgrowth41,42, and granule cell survival243, which could potentially influence the development of the cerebellar cortex, and correlational studies have found deficiencies in cerebellar-associated behavior in humans whose mothers consumed cannabis during pregnancy6,189. Additionally, exogenous cannabinoid exposure in pregnant rodents affect cerebellar circuitry and cerebellar-dependent behaviors after birth, but only a few have examined exogenous cannabinoid influences on behavioral phenotypic abnormalities during an age in a third trimester human fetus equivalent rat model190,204. In this Chapter, we exposed PND 2-10 rats to either saline or the exogenous CB1R agonist WIN 55,212-2 (WIN) and examined the resulting effects on spontaneous granule cell afferent synaptic transmission at PND 10 and PND 30-35. We also determined impacts of PND 2-

10 rat WIN exposure on accelerating rotarod proficiency, a behavioral task thought to detect

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cerebellar signaling deficiencies, at PND 30-40. We found negligible impacts on granule cell afferent synaptic transmission, but observed that WIN-exposed rats had difficulty navigating the accelerated rotarod behavioral task. These results suggest that exogenous cannabinoid exposure in a third trimester equivalent rodent model may negatively disrupt cerebellar development, which have long-term effects on motor coordination. Although we only measured a small component of cerebellar processing electrophysiologically, our data finds no compelling differences between saline and WIN treatment groups. This may indicate cerebellar signaling is disrupted elsewhere other than mossy fiber->GC and Golgi cell->GC synapses, or rotarod deficiencies are due to cellular alterations beyond the cerebellum.

INTRODUCTION

Based on knowledge implicating endocannabinoids as important neurodevelopmental signaling molecules and the developmental CB1R-mediated reduction in polysynaptic glutamate release onto Purkinje cells and migrating granule cells, we explored whether synthetic cannabinoid exposure in third-trimester equivalent rat pups could impact cerebellar development. Since this developmental time window is arguably the most important for proper cerebellar maturation, and is a period in which pregnant mothers often consume cannabinoid products4, we were interested in the effects of exogenous cannabinoids on the outcome of various developmentally- sensitive phenotypic endpoints, such as neuronal signaling and cerebellar-dependent behaviors.

An estimated 3-5% of American women consume cannabis during third trimester pregnancy3,6. Although the results of human longitudinal studies have been mixed, previous literature indicates human prenatal exposure to cannabis correlated with impairments in

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executive function198, reading/writing comprehension199, motor development189, and motor coordination6. Prenatally exposing rats to cannabinoids alters cortical neuronal migration193,

Purkinje cell firing68,244, long term depression196, memory retention187, and producing motor hyperactivity in offspring187,203. A large proportion of these results have been mixed due to differences in various study protocols, such as the cannabinoid used, the time and frequency of exposure, route of administration, and age of testing205. For example, many of these studies have determined the extent of behavioral deficiencies produced in offspring of pregnant rodents exposed to cannabinoids, but very few studies have examined the effects of cannabinoid exposure on postnatal animals. This is relevant because this rodent postnatal timeframe is a better model for examining impacts of drugs on cerebellar development during the most significant period of cerebellar growth. Recently there has been some work implicating rat PND

4-9 synthetic cannabinoid exposure on early motor development deficiencies204, which provides grounds to suggest exogenous cannabinoids may disrupt cues vital to proper neurodevelopment of the cerebellar cortex.

There are a number of developmental stages in which neurons rely on molecular cues to signal cessation of a particular process. Mature cerebellar granule cells have approximately four dendrites with claw-like extremities that synapse onto mossy fiber and Golgi cell axons to form granule cell layer glomeruli. The number of granule cell dendrites typically prune back from approximately 8 to 4 following maturation, in part due to receptor activation and intracellular signaling, at least partially due to glutamatergic signaling21. Because dendritic pruning relies on neuromodulatory processes involved in development, altering these processes, whether it be

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directly through glutamate transduction or indirectly via cannabinoid influences on glutamate release, may potentially alter the cues used to determine overall dendritic survivability.

To our knowledge there is a gap in understanding how CB1R-induced developmental and cellular disruption of neural circuits leads to cerebellar behavioral deficits. None have attempted to examine the role of cannabinoids in modulating transsynaptic glutamatergic signaling, a modulatory process involved in coordinating and refining neuronal migration and synaptogenesis as a mechanism to sculpt maturing tissue245. However, a related study determined maternal cannabinoid exposure can alter Purkinje cell membrane excitability and cerebellar-dependent behavioral output in offspring68, which may provide clues into how CB1R-mediatied glutamate reduction can modify the electrophysiological properties of granule cells. Recording from granule cells following cannabinoid exposure may enable us to determine whether there are altered synaptic properties that change granule cell excitability, such as changes in afferent synaptic release from mossy fibers and Golgi cells.

In the current chapter we expand the findings within previous chapters to determine whether in vivo cannabinoid exposure produces cellular abnormalities and alterations in cerebellar-dependent behavioral phenotypes. In order to determine whether there are impacts of exogenous cannabinoid exposure on aspects of cerebellar development, we injected rats with saline (vehicle) or the CB1R agonist WIN 55,212-2. We then characterized differences in synaptic signaling and cerebellar-dependent behaviors, thereby facilitating comparisons between how cellular actions of exogenous cannabinoids may affect cerebellar processing acutely in rat pups and chronically/long-term weeks after exposure.

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Besides our previous data finding a pronounced reduction in migrating granule cell

NMDAR activation following cannabinoid application, we hypothesize exposure of a CB1R agonist during this period will markedly alter cerebellar development of granule cells, disrupting typical cerebellar signal processing, and manifesting as altered cerebellar-dependent behavioral phenotypes.

METHODS

Animals. All animals were bred and housed within a Washington State University (WSU) vivarium.

All procedures conformed to regulations approved by the Washington State University

Institutional Animal Care and Use Committee (IACUC), and conformed to all guidelines for ethical protocols and care of experimental animals established by the National Institutes of Health,

Maryland, USA. Sprague Dawley rats (Simonsen) were used for all experiments.

Injections. Starting at PND 2, pups received (between 9am-11am) subcutaneous injections of either CB1R agonist WIN 55,212-2 (0.5 mg/kg or 3mg/kg) or vehicle daily from PND 2 to PND 10.

Drug concentration was identical for both WIN treatment groups, with the only difference being volume used (3X greater volume for 3mg/kg rat pups). Accordingly, there were two saline treatments: one lower volume and one higher volume. Both vehicle and WIN solutions contained

1% of the vehicle detergent TWEEN 80 (polysorbate 80). Injections were 6µl per gram weight.

Treatments alternated for each new litter born to best control for extraneous environmental factors. Pups were left with the mother and allowed to breastfeed as usual. Males and females were weaned and separated at PND 21.

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MgCl2, and 10 mM D-glucose. All aCSF was bubbled with 95% O2/5% CO2 and contained 1 mM kynurenic acid (a glutamate receptor antagonist used to prevent potential excitotoxicity within the tissue). The cerebellum was microdissected out and parasagittal slices (225 µm) of the cerebellar vermis were taken using a Leica VT1200S vibratome in a slicing chamber filled with bubbling 1°C aCSF. Slices were then incubated in aCSF containing 1 mM kynurenic acid at physiological temperature (34-35°C) for 1 hour before electrophysiological recordings were conducted.

Slice Electrophysiology. Slices were secured with a platinum harp in a submersion chamber mounted on an Olympus BX51WI microscope, and visualized with a 60X (0.90 N.A.) water- immersion objective. Slices were perfused at a rate of 5-7 ml/min with artificial cerebrospinal fluid (aCSF), maintained at a temperature between 32-36° C, and bubbled with a 95% O2/5% CO2 gas. Granule cells were visually identified and voltage-clamped (Vh = -30mV) with patch electrodes made from borosilicate glass capillary pipettes. Pipettes contained: 130 mM

CsGluconate, 4 mM NaCl, 0.5 mM CaCl2, 10 mM HEPES, 5 mM EGTA, 4 mM MgATP, 0.5 mM

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Na2GTP, and 5 mM QX-314. Internal solution was pH-adjusted to 7.2-7.3 with CsOH. Electrode resistance was 4-8 MΩ for granule cells. Cells were excluded if access resistance changed by >20%

- throughout the duration of an experiment. Given the extra- and intracellular [Cl ], ECl was ~ -

60mV, so voltage-clamping at -30mV resulted in IPSCs being outward and EPSCs being inward. All recordings were filtered at 10 kHz and acquired at 20kHz. Drugs were dissolved in aCSF and constantly bubbled with 95% O2/5% CO2 gas before being administered. All experimental conditions were conducted for 5 minutes each, with the baseline condition being conducted for at least 5 minutes or until a stable baseline was reached.

Analysis of spontaneous and EPSC/IPSC currents. Spontaneous synaptic events (sIPSCs & sEPSCs) from the final two minutes of recording in each experimental condition were analyzed using Mini

Analysis 6.0.7 (Synaptosoft). Automatic detection of sIPSCs and sEPSCs was executed, using an amplitude threshold of 2 times the peak to peak amplitude of the noise, and then events were individually inspected with a further inclusion criterion of having a rise time at least 3 times faster than the decay time. Average frequency was determined including temporally overlapping events, then all non-overlapping events were averaged to calculate mean amplitude. All cells with lower than 0.1 Hz spontaneous baseline frequency were excluded from analysis. Because the

CB1R agonist WIN 55,212-2 fails to wash out within a reasonable amount of time, the wash condition is excluded in the following results unless noted otherwise.

Newborn rodent Injections. Sprague Dawley rat litters were culled down to six pups per litter

(3M/3F) at PND 2 and pups subsequently received (between 9am-12pm) subcutaneous

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injections of either CB1R agonist WIN 55,212-2 (3mg/kg) or saline daily from PND 2 to PND 10.

Both saline and WIN solutions contained 1% of the vehicle detergent TWEEN 80 (polysorbate

80). Injections were either 1µl per gram weight for the 0.5mg/ml WIN condition and its associated saline control group, or 6µl per gram weight for the 3.0mg/kg WIN condition and its associated saline group. Injection volume differed according to drug treatment group (and associated saline conditions) because the same concentration of WIN solution was used. Pups were left with the mother and allowed to breastfeed as usual. Males and females were weaned and separated at PND 21.

Accelerated Rotarod. PND 30 rats (that had been injected with WIN or saline as pups) were allowed a single training day with two training sessions (Training 1 & 2) in which they were given

8 sessions to learn to navigate an accelerating rotarod in which a rotating rod (0-20 seconds: fixed

7 RPM; 20-180 seconds: linearly accelerated from 7-50 RPM) continually accelerates until each rat fell. Latency to fall was recorded (in seconds). Following the initial training day, rats were then subjected to 10 daily 5-session trials (Trial 1-10) until PND 40.

Statistics. Data was compared by analyzing the means for each condition ± SEM.

Electrophysiology experiments were analyzed using two-way analysis of variance (ANOVA), and rotarod experiments were measured using repeated measures two-way ANOVA. For electrophysiology experiments, Treatment (either Saline or WIN injections) and Condition (either baseline sPSC frequency or 5µM WIN application) were compared. One-way analysis of variance

(ANOVA) was used to compare mean baseline holding currents between PND 30-35 granule cells.

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For rotarod, injection Treatment and Testing Day were compared. Tukey’s pairwise multiple comparison tests were performed for all main effects to determine differences between treatments. All statistical measures were made using SigmaPlot 11.0 software (Systat Software,

Inc).

Reagents. Kynurenic acid (1mM; Abcam; ab120064) was added to aCSF solution during brain extraction. The CB1 agonist (+)-WIN 55,212-2 was purchased from Cayman Chemical (Cat. No.

10009023). Tween 80 was purchased from Sigma Aldrich (P1754), and 0.9% sodium chloride solution (saline) was used for injections (Hospira; RL-4492). The vehicle solution contained 1%

Tween 80, which was used in Saline and WIN treatment conditions.

RESULTS

Electrophysiology

All electrophysiological recordings were whole-cell voltage-clamp recordings (Vh=-30mV; ECl=-

60mV) of granule cells, as described in the Methods section. We recorded baseline levels of sPSCs before bath applying 5µM WIN, which represents ‘condition’. ‘Treatment’ refers to drug treatment received during injections. Since we used the same WIN concentration but administered different volumes between 0.5 and 3mg/kg drug treatments, we had two control saline treatment groups. The low-volume saline group received the same volume of vehicle solution as the 0.5mg/kg WIN treatment group, whereas the high-volume saline group received the same volume of vehicle solution as the 3mg/kg WIN treatment group. Previous studies have found disrupted cerebellar function in Purkinje cells following WIN injections68,191. Since we see

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disrupted glutamatergic signaling in developing brains following 5µM WIN administration in slice, and coupled with knowledge of the importance of glutamate in brain processes such as migration and synaptogenesis, we predict granule cell synaptic transmission will be disrupted as well. This may due to altered migrational glutamate cues following WIN exposure, leading to improper positioning with the granule cell layer, or impacted glutamate-mediated synaptogenesis.

Developing tissue (PND 10)

PND 10 sEPSC FREQUENCY

When comparing sEPSC frequency between groups, two-way analysis of variance (ANOVA) revealed there was no main effect (F(3,87)=0.353, p=0.787) between ‘treatment’ (F(3,87)=0.934, p=0.428) or ‘condition,’ but there was a significant difference between ‘condition’ levels

(F(1,87)=4.225, p=0.043) (Fig. 4.1A). Tukey’s multiple comparisons found there a difference in sEPSC frequency between the baseline and 5µM WIN conditions within the 0.5mg/ml treatment group (p=0.040). (Fig. 4.1A). The general lack of WIN effect is surprising considering our previous findings showing strong reductions in synaptic transmission following WIN application. There may be other potential effects reducing the effectiveness of WIN, which we discuss this later in the chapter.

PND 10 sEPSC AMPLITUDE

When comparing sEPSC amplitude between groups, two-way analysis of variance (ANOVA) revealed there was no main effect (F(3,93)=0.162, p=0.922) between ‘condition’ (F(1,93)=0.033, p=0.856) or ‘treatment,’ but there was a significant difference between ‘treatment’ levels

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(F(3,93)=4.363, p=0.006) (Fig. 4.1A). Tukey’s multiple comparisons found there was no differences between treatments during individual recording conditions. sEPSC amplitude was significantly different between the 0.5mg/kg WIN and high-volume saline treatment groups when comparing using the ‘treatment’ factor (summed sIPSC amplitudes in both baseline and

5µM WIN conditions for both treatments were compared) (p=0.009) (Fig. 4.1B). The lack of difference between baseline sEPSC amplitudes across treatment groups may imply there are no differences in postsynaptic modulation such as AMPAR density of expression and sensitivity.

PND 10 sIPSC FREQUENCY

When comparing sIPSC frequency between groups, two-way analysis of variance (ANOVA) revealed there was no main effect (F(3,117)=0.154, p=0.927) between ‘treatment’ and

‘condition,’ and both ‘treatment’ (F(3,117)=6.392, p<0.001) and ‘condition’ (F(1,117)=10.670, p=0.001) show significant differences in their mean sIPSC frequencies (Fig. 4.2). Tukey’s multiple comparisons shows that sIPSC frequencies differ in the baseline condition between the 0.5mg/kg

WIN group and the high-volume saline group (p=0.034) (Fig. 4.2A). The sIPSC frequency of the

3mg/kg WIN treatment group was significantly reduced following 5µM WIN application (p=0.018)

(Fig. 4.2A). Again, a general lack of WIN effect on sIPSC frequency was surprising given our previous results. If both sEPSC and sIPSC frequencies are resistant to WIN effects in WIN and non-

WIN treated rats, it may imply there is an external neuromodulatory effect on synaptic transmission, such as a stress effect on CB1R expression Although not significant, the lower- volume saline and lower volume (0.5mg/kg) WIN groups appear to display lower baseline sIPSC frequency levels, which is interesting considering the only difference is injection volume.

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PND 10 sIPSC AMPLITUDE

When comparing sIPSC amplitude between groups, two-way analysis of variance (ANOVA) revealed there was no main effect (F(3,118)=0.0761, p=0.973) between ‘condition’

(F(1,118)=2.627, p=0.108) or ‘treatment,’ but ‘treatment’ significantly differed between levels

(F(3,118)=3.433, p=0.019) (Fig. 4.2B). Although no individual differences were found, Tukey’s multiple comparisons found sIPSC amplitude was significantly different between the 0.5mg/kg

WIN and high-volume saline treatment groups when comparing using the ‘treatment’ factor

(summed sIPSC amplitudes in both baseline and 5µM WIN conditions for both treatments were compared) (p=0.029) (Fig. 4.2B). The lack of change in sIPSC amplitude following WIN application was no surprising given our there was little difference in sIPSC frequency, and our previous results have failed to see a difference in amplitude.

Mature tissue (PND 30-35)

In addition to examining whether exogenous cannabinoid exposure during cerebellar development may have neurophysiological impacts while still in development, we sought to determine whether impacts could persist into adolescence/adulthood. Rather than recording from PND 10 granule cells following PND 2-10 WIN injections, rats were allowed to mature following injections unto PND 30 when recordings were carried out. We measured granule cell sPSC frequencies and amplitudes before and after 5µM WIN application.

PND 30-35 sEPSC FREQUENCY

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When comparing sEPSC frequency between groups, two-way analysis of variance (ANOVA) revealed there was no main effect (F(1,56)=0.016, p=0.90) between ‘treatment’ (F(1,56)=0.0751, p=0.785) or ‘condition’ (F(1,56)=0.0685, p=0.794) factors (Fig. 4.3A). There were no changes in sEPSC frequency following WIN application, which agrees with our previous findings in mature mossy fiber to granule cell synapses. Finding a difference in sIPSC frequency between treatment groups may have suggested the effects of PND 2-10 WIN treatment are long-lasting in granule cell synaptic function.

PND 30-35 sEPSC AMPLITUDE

When comparing sEPSC amplitude between groups, two-way analysis of variance (ANOVA) revealed there was no main effect (F(3,166)=0.177, p=0.912) between ‘treatment’

(F(3,166)=1.497, p=0.217) or ‘condition’ (F(1,166)=0.763, p=0.384) factors (Fig. 4.3B). A null result suggests WIN-exposure may not have effected postsynaptic receptor modulation.

PND 30-35 sIPSC FREQUENCY

When comparing sIPSC frequency between groups, two-way analysis of variance (ANOVA) revealed there was no main effect (F(3,66)=0.113, p=0.952) between ‘treatment’ (F(3,66)=0.782, p=0.508) or ‘condition,’ but there was a significant difference between levels in the ‘condition’ factor (F(1,66)=10.597, p=0.002) (Fig. 4.4A). Although no individual differences were found,

Tukey’s multiple comparisons found sIPSC amplitude was significantly different between baseline and 5µM WIN conditions when comparing using the ‘condition’ factor (summed sIPSC amplitudes of all treatment groups between both baseline and 5µM WIN conditions were compared)

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(p=0.002) (Fig. 4.4A). Our previous data in mature Golgi to granule cell synapses has indicated a strong WIN-induced synaptic suppression of vesicle release. While there appears to be a general trend toward reduced sIPSC frequency in all treatment groups, the lack of individual differences is surprising.

PND 30-35 sIPSC AMPLITUDE

When comparing sIPSC amplitude between groups, two-way analysis of variance (ANOVA) revealed there was no main effect (F(3,65)=0.386, p=0.763) between ‘treatment’ and ‘condition.’

However, the difference in mean sIPSC amplitudes among different ‘treatment’ groups

(F(3,65)=2.853, p=0.044) and between ‘conditions’ (F(1,65)=5.264, p=0.025) significantly differed

(Fig. 4.4B). Although no individual differences were found, Tukey’s multiple comparisons found sIPSC amplitude was significantly different between baseline and 5µM WIN conditions when comparing using the ‘condition’ factor (summed sIPSC amplitudes of all treatment groups between both baseline and 5µM WIN conditions were compared) (p=0.025) (Fig. 4.4B). Similar to the sIPSC frequency, there is a general reduction in sIPSC amplitude following WIN application.

This is interesting because we see a reduction in sIPSC amplitude in our previous findings in mature granule cells, which may imply some form of synaptic modulation following CB1R activation.

PND 30-35 Holding Current

Granule cells in mature rats (PND 38) are observed to undergo tonic GABAergic inhibition through activation of GABAARs, a result of GABA accumulation in the Golgi/granule synapse246.

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Differences in the tonic inhibition of granule cells in mature tissue may provide insight into whether cannabinoid exposure can alter aspects of Golgi/granule cell synaptic properties. For example, differences in an overall tonic inhibition may imply either increased extrasynaptic

GABAAR receptor expression (increased number of tonic postsynaptic receptor activation), or increased Golgi cell to granule cell synaptic contact (potential increase in GABA concentration able to spill out of the synapse), etc. We compared the baseline holding current while recording from granule cells (Vh=-30mV; ECl=-60mV) to determine whether PND 2-10 WIN exposure may have influenced synaptic modulators controlling tonic GABAergic granule cell inhibition. One-way

ANOVA revealed there were no differences in the baseline holding current between treatment groups, F(3,83)=0.322, p=0.809) (Fig. 4.5). These data seem to suggest that WIN exposure may not impact extrasynaptic GABAergic inhibition of granule cells, since changes in extrasynaptic

GABAAR expression and sensitivity would likely explain a change in tonic inhibitory holding current without changes in sIPSC characteristics.

Accelerated Rotarod

Rat pups that underwent single daily (PND 2-10) subcutaneous injections of either saline or

3mg/kg WIN 55,212-2 were subjected to a daily accelerated rotarod behavioral task from PND

30-40 (Fig. 4.6A). Two-way analysis of variance (ANOVA) revealed a main effect (F(11,377)=3.00, p<0.001) between ‘treatment’ (F(1,48) = 4.3, p<0.05) and ‘testing day,’ (F(11,377) = 18.8, p<0.001). Tukey’s multiple comparisons found there were no significant differences between treatment groups during the initial training phases (Training 1 & 2), in which rats learned to walk on the rotating rod, but there were treatment differences in fall latency on the first four testing

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days (Trial 1-4): % difference in fall latency between drug treatment groups: Trial 1 = 38.8

(p<0.01); Trial 2 = 36.7 (p<0.01); Trial 3 = 28.1 (p<0.05); Trial 4 = 27.6 (p<0.05) (Fig. 4.6E). There were no significant differences in rat weights between treatment groups, either during the injection period (PND 2-10; Fig. 4.6B) or during the rotarod task (PND 30-40; Fig. 4.6C). The initial rotarod performance deficiencies in WIN-treated rats suggests there may be an impact on motor learning between the two treatment groups. This is further supported by the data showing WIN- exposed rats eventually display similar fall latencies by Trial 5 as saline rats. If motor learning has been impacted, it may indicate a disruption in cerebellar processing in rats exposed to WIN during cerebellar development.

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FIGURE 4.1 A

Figure 4.1. PND 10 granule cell sEPSC frequency and amplitude in PND 2-10 drug-treated rats. (A) The sEPSC frequency is reduced only in the 0.5mg/kg WIN treatment group following 5µM WIN application (p=0.04). (B) There were no individual differences in sEPSC amplitudes between treatment groups or conditions. However, the 0.5mg/kg WIN and high-volume saline treatment groups differed when grouping both baseline and WIN conditions together (p=0.009). * indicates p<0.05

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FIGURE 4.2 A

Figure 4.2. PND 10 granule cell sIPSC frequency and amplitude in PND 2-10 drug-treated rats. (A) The sIPSC frequency is reduced only in the 3mg/kg WIN treatment group following 5µM WIN application (p=0.018). The baseline sIPSC frequencies between the 0.5mg/kg WIN and high-volume saline group were significantly different (p=0.034). (B) There were no individual differences in sIPSC amplitudes between treatment groups or conditions. However, sIPSC amplitudes between the 0.5mg/kg WIN and high-volume saline treatment groups differed when comparing them using both baseline and 5µM conditions (p=0.029). * indicates p<0.05

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FIGURE 4.3 A

Figure 4.3. PND 30-35 granule cell sEPSC frequency and amplitude in PND 2-10 drug-treated rats. (A) sEPSC frequency did not differ between treatment groups or between conditions (p>0.05). (B) There were no differences in sEPSC amplitudes between treatment groups or conditions (p>0.05).

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FIGURE 4.4 A

Figure 4.4. PND 30-35 granule cell sIPSC frequency and amplitude in PND 2-10 drug-treated rats. (A) The overall sIPSC frequency in the 5µM WIN condition were significantly less than baseline frequencies when comparing all treatment groups together (p=0.002). There are no individual differences between treatment groups or conditions. (B) Similar to sIPSC frequency, the overall sIPSC amplitude in the 5µM WIN condition were significantly less than baseline frequencies when comparing all treatment groups together (p=0.025). There were no individual differences between treatment groups or conditions.

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FIGURE 4.5

Figure 4.5. PND 30-35 granule cell mean baseline holding current. Whole-cell patch clamped granule cells (Vh = -30mV; ECl = -60mV) generally exhibited a negative holding current, indicating slight depolarization. There were no significant differences in baseline granule cell holding current between treatment groups (p=0.809), which may suggest neither GABAergic Golgi innervation or granule cell GABAAR expression is affected by our manipulations. However, there may be a trend toward greater hyperpolarization in WIN-treated animals.

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FIGURE 4.6

Figure 4.6. Rat pups exposed to CB1R agonist WIN 55,212-2 PND 2-10 found to exhibit motor deficiencies during PND 30-40 accelerated rotarod behavioral task. (A) A timeline depicting the injection period, in which rat pups received daily subcutaneous injections of either vehicle (saline) or 3mg/kg WIN. Once pups reached PND 30, they were subjected to a daily accelerated rotarod task until PND 40. (B) Rat pups did not differ in weight between treatment groups. (C) Rats subjected to the rotarod task did not differ in weight. (D) A series of example images showing a rat navigating the rotarod task before falling, in which latency to fall is recorded. (E) Following a rotarod training period, rats exposed to WIN exhibited deficiencies in navigating the rotarod task when compared with vehicle treated pups, exhibiting a reduced latency before falling from the rod. WIN treated rats reached similar rotarod proficiency to the vehicle-treated group by Trial 5, which may indicate an initial deficit in cerebellar motor learning. * indicates p<0.05; ** indicates p<0.01

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FIGURE 4.7 A

Figure 4.7. Rotarod performance broken down into both initial Training sessions. No significant differences between treatment groups appeared during either Training 1 (A) or Training 2 (B) sessions.

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TABLE 4.1

Table 4.1. Average latency to fall during the accelerated rotarod behavioral task. Rats treated with the CB1R agonist 3mg/kg WIN as pups (PND 2-10) performed significantly worse at the cerebellar-dependent rotarod task during the first four days (Trial 1-4). WIN-treated rats then reached similar proficiency levels as vehicle (saline) treated rats, suggesting the CB1R activation during development interferes with motor learning circuitry, leading to long-lasting behavioral deficiencies. * indicates p<0.05 ** indicates p<0.01

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TABLE 4.2

Table 4.2. Average rat pup weight before each injection. A table displaying numerical values for rat weights. There were no significant differences in weights between treatment groups. P>0.05

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TABLE 4.3

Table 4.3. Average rat weight before each rotarod session. A table displaying numerical values for rat weights. There were no significant differences in weights between treatment groups. P>0.05

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DISCUSSION

The data from experiments contained within this chapter attempt to recreate the conditions that produced the cannabinoid effects of the previous two chapters in an in vivo model. We initially began injection experiments using the lower concentration of WIN (0.5 mg/kg), as prior publications have indicated this a satisfactory dose to prevent toxicity or gross malformations but still leading to cellular and behavioral deficits194. There was no significant decrease in sEPSC frequency following WIN application in either saline/vehicle-treatment condition (Fig. 4.1A).

Finding no significant differences in granule cell sIPSC frequency following this dose either, we happened upon a study examining the brain levels of WIN following specific doses of WIN intraperitoneal injections in 28-30 g mice (which correlates to roughly PND 35, according to a developmental growth chart (Charles River Laboratories))247. Approximately 0.3% of total injected WIN was found in brain tissue in a dose dependent manner, with less than 0.5 nmol/g found following 0.3 mg/kg WIN injections. This was below the level of detectable response in

Purkinje cells according to a previous dose-response curve developed for Purkinje CB1R activation163. The 3 mg/kg dose of WIN produced approximately 0.8 nmol/g WIN accumulation, which is closer to the saturating dose used for max CB1R activation levels in Purkinje cells163, which is why we chose this higher dose in subsequent injection experiments.

Despite appropriating the WIN dosage, our only observed differences between treatment groups was between baseline PND 10 GABAergic sIPSC frequencies in the 0.5mg/kg WIN and high-volume saline groups (Fig. 4.2A). Of course, this difference is more difficult to attribute to changes in experimental conditions given that the data compared is a between-subject design, making differences in sPSC frequency more variable. We attempted to alternate treatment

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conditions for every rat litter born in order to equalize any potential environmental factors that may have played a role in experimental variability, yet experiments using the high vehicle/WIN treatment groups were performed at a later time period, and a higher injection volume which, although nominal, may have been stress inducing.

The most puzzling result stemmed from the lack of WIN effect on PND 10 granule cell sEPSC frequencies in three of the four treatment groups. Only the 0.5mg/kg WIN treatment group seemed to respond to WIN (Fig. 4.1A), which is surprising considering the higher 3mg/kg WIN treatment (with a greater n size, 19) did not. This leads us to speculate whether there may be external factors at play. Our first inclination would be to suspect residual WIN effects, such as general synaptic suppression, due to injections earlier that day (final injection at ~9am that morning). However, WIN also failed to reduce granule cell sEPSC frequency in both saline treatment groups as well, which makes this possibility unlikely. As we briefly mentioned, stress may be a more probable factor explaining our lack of WIN effect; both chronic unpredictable mild stress (CUMS) and chronic unpredictable stress (CUS) have been shown to reduce hippocampal

CB1R binding density248,249, whereas chronic corticosterone exposure in the amygdala and hippocampus250. If similar mechanisms exist within the cerebellum, stress from handling and injecting rat pups over multiple days could reduce CB1R expression onto granule cells, which would thus reduce CB1R-activated vesicle reduction following 5µM WIN administration. Of course, it could be argued that our analysis is simply underpowered. For example, our original granule cell sEPSC (n=10) recordings had more cell numbers than the PND 10 sEPSC low-volume saline sEPSC recordings (n=5). However, that is less than the 0.5mg/kg WIN (n=12), high-volume saline (n=11), and 3mg/kg WIN (n=19) treatment groups. This argument may make more sense

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for our PND 10 sIPSC frequency analysis since they also had a general lack of WIN effect. In this case our original granule cell sIPSC (n=19) recordings had slightly larger cell numbers than the

PND 10 sIPSC low-volume saline (n=15), 0.5mg/kg WIN (n=14), and high-volume saline (n=15), but equivalent to the 3mg/kg WIN treatment group (n=20), which also happened to show a significant reduction in sIPSC frequency in 5µM WIN. Each case indicates gathering additional data may be important before solid conclusions can be made.

We also examined the mean baseline granule cell voltage-clamp holding currents between different treatment groups as an indirect measure of tonic GABAAR-induced inhibitory current, as GABAergic spillover and resulting extrasynaptic GABA activation of GABAARs in granule cell synapses are known to lead to tonic granule cell inhibition, and control overall information flow through the cerebellar cortex246,251,252. Typically a more effective measure of tonic GABAAR current is to measure differences in holding current following the application of a

253 GABAAR blocker such as Gabazine . However, since we were subjecting all cells to WIN, which is very difficult to wash out, we were unable to appropriately measure tonic current with

Gabazine, considering CB1R activation may influence this property. As a consolation, we compared baseline holding current since the holding current should reflect intrinsic GABAAR activity. The mean baseline current of granule cells did not significantly differ between treatment groups (Fig. 4.5), which may either indicate there are likely no differences in ambient GABA levels or a change in extrasynaptic GABAAR expression, or the statistical analysis is underpowered. It is possible there is a trend with WIN-exposed treatment groups approaching a more depolarized state, but there may need to be more recordings to determine this with confidence. If WIN-

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treated granule cells did exhibit relatively less hyperpolarization, granule cell excitability would be enhanced and potentially lead to inappropriate granule cell action potential generation.

Interestingly, our data showed WIN-treated rats performed significantly poorer on the accelerated rotarod behavioral task when compared to vehicle treated rats. Notably, these differences failed to manifest until after the initial training day, with WIN-treated rats falling from the rotarod significantly sooner on the first four testing days than vehicle-treated rats before reaching similar proficiency levels. The first training period was comprised of eight individual sessions, and when observing these individually, there appears to be little difference between treatment groups, as with the second training period (Fig. 4.8). So according to the results of the first four testing days it appears WIN injections may have interfered with a component of cerebellar-dependent motor learning, as the accelerating rotarod paradigm is known as an established paradigm for motor skill learning to measure short- and long-term improvements254.

Finding a difference in rotarod proficiency between treatment groups despite no differences seen in granule cell activity appears counterintuitive. Despite PND 30-40 vehicle and

WIN treated rats showing similar levels of baseline glutamatergic and GABAergic input, as well as similar effects in response to WIN application in slice, the rotarod deficiency in WIN-treated animals was fairly robust. The few electrophysiological differences observed between treatment groups may be indicative of the narrow focus we have employed; simply attempting to observe a change in synaptic frequency onto granule cells may result in painting an incomplete picture in terms of the various indirect mechanistic effects CB1R activation may have on cerebellar development.

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The discrepancy between our rotarod and granule cell electrophysiological results could potentially be explained by a developmental modification separate from granule cells, perhaps downstream. For example, to address a few developmental studies, recent evidence highlights mechanisms underlying climbing fiber synaptic pruning37, which are dependent on proper granule cell innervation255 and presumably endocannabinoid modulation. As previously noted, climbing fibers generally prune back synaptic innervation from multiple Purkinje cells to a single

Purkinje cell37, and it is thought that mature rats lacking granule cells maintain multiple climbing fiber innervations256. Therefore, it is possible that glutamatergic innervation by parallel fibers is able to shape climbing fiber synapses onto Purkinje cells, and thereby influencing the ability of the cerebellum to relay motor error signals20. CB1R expression has been found in mature climbing fiber axons, and it is possible that climbing fibers express CB1Rs at an earlier age as well (PND 9-

14257). Further studies would be enlightening to determine whether climbing fibers express

CB1Rs during development, if a CB1R agonist can alter climbing fiber synaptic transmission onto

Purkinje cells, and whether WIN injections at PND 2-10 could affect the number and complexity of climbing fiber synaptic contacts with Purkinje cells. Also, Shabani et al. (2011) found differences in Purkinje cell excitability following maternal WIN exposure, suggesting modulation of intrinsic ion channels, and a later study concluded WIN exposure enhanced Ca2+ channel current amplitude244. Although exposed at a different developmental time period, these results may aid in shaping our understanding of WIN effects in developing cerebellar tissue.

Additionally, several studies have seen a correlation between neuronal polarization and commencement of long-range cell migration with early CB1R expression235,258. There are distinct

CB1R-expressing hippocampal GABAergic interneurons neurons that follow their own complex

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migratory path, and establish early synapses before migration is completed. Also, although in embryo, CB1Rs have been found to promote axonal growth. In this way exogenous CB1R activation may oppose, or counteract, the effects of reduced glutamatergic signaling onto migrating granule cells; the former promotes migration while the latter opposes migration. CB1Rs are also thought to be involved in neuronal commitment, as their expression coincides with differentiation in chick embryo neurons259. Again, despite being embryos, there may be parallel forms of development we can extrapolate as a potential source of developmental disruption.

These examples provide alternative explanations for the lack of significant granule cell synaptic property differences following cannabinoid treatment, which is contrary to the discrepancies in rotarod proficiency.

A final alternative explanation for WIN-induced rotarod deficiency could be developmental disruption in a brain region other than the cerebellar cortex. The CB1R agonist

WIN—a drug active at ubiquitous CB1/2Rs throughout the central and peripheral nervous systems—was administered systemically, and presumably affects the development of multiple brain regions, which may include other regions involved in motor skills. As mentioned in the introduction, other studies have seen alterations in behavioral effects such as impaired emotional reactivity tasks, probabilistic reward choice tasks, and memory retention in addition to motor incoordination following WIN administration187,194,195. So for our work it is difficult to rule out other motor regions involved in motor coordination, especially after a lack of granule cell differences in afferent transmission between treatment groups. In addition to the cerebellum, the basal ganglia is thought to be intimately involved in motor learning260, and previous groups have found striatal CB1R activation is linked to motor deficits89. In fact, the

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endocannabinoid system has been implicated in influencing aspects of motor control within the striatum, and drugs blocking the degradation of the endocannabinoid 2-AG can rescue motor deficits seen within a mouse Parkinson’s model261. This links cannabinoid receptor activation and striatal motor pathways and shows how both the endocannabinoid system and basal ganglia can impact motor control outside the cerebellum. Thus, it is likely that systemic WIN exposure has affected the development of numerous brain regions besides the cerebellum, and focused studies may be required to segregate the behavioral effects that accompany gross systemic CB1R activation with specific brain region dysfunction.

Additionally, it is likely that no single mechanism completely accounts for the differences observed in rotarod proficiency following WIN exposure, but rather, a combination of molecular and cellular phenotypic factors. To address this, there are an exhaustive number of measures that could be used to determine differences in cerebellar morphology and function following cannabinoid exposure during development. We could measure electrophysiological and synaptic properties of Purkinje cells, since Purkinje cell dendritic spines increase following learning complex motor skills262. We could also examine whether there are differences in gross cerebellar morphology such as layer size and proportions of post-migratory cells. Changes in these attributes may suggest population differences in granule cells able to participate in network function, as granule cells have been found to exhibit anticipatory activity to eyeblink conditioning in mice263, which shows clear motor learning. We could also measure quantitative protein expression, which we could hypothesize to be affected by inappropriate developmental cannabinoid activation.

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Conclusions

The previous two chapters have 1) revealed the transient functional expression of CB1Rs in specific cerebellar synapses and 2) established the ability of upstream CB1R activation to dampen glutamatergic guidance cues onto Purkinje cells and migrating granule cells. As a culmination of these two chapters, the results of this chapter demonstrate that exposure to an exogenous cannabinoid during a critical cerebellar developmental period has prolonged consequences on cerebellar dependent behaviors at a later age. Our electrophysiological results in WIN-treated granule cells show no significant differences between treatment groups, which may be initially surprising considering our rotarod data, but may elude to cellular disruptions elsewhere within the cerebellar cortex, such as Purkinje cells or their climbing fiber afferents.

Because of the current rapid legalization of cannabis, and its subsequent perceived innocuous nature, it is important to concurrently establish an understanding of how exogenous cannabinoids could change cerebellar function during development. In addition, using the endogenous endocannabinoid system within the cerebellar cortex as a model system helps us understand how cannabinoids may influence synaptic transmission as well as how endocannabinoids could alter acute neurodevelopmental functioning in general. In previous chapters, our most clear finding is that CB1R activation affects many aspects of cerebellar signaling during development. Although most of our electrophysiological findings in granule cells appear unchanged following exogenous cannabinoid exposure, the primary finding of this chapter is quite clear: exogenous cannabinoid exposure during the most developmentally sensitive time period of cerebellar growth produces noticeable motor deficits in rats, which may require additional work to determine if these motor deficits are cerebellar in origin.

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CHAPTER 5

General Discussion

Overview

The scientific community has made considerable advances in understanding the function of the endocannabinoid system within the mammalian brain. This includes elements of understanding the role that cannabinoids play as neurodevelopmental signaling lipids and encompassing aspects of modulatory control in neuronal migration, axonal growth, and synaptogenesis87.

However, many of these advances have been piecemeal; researchers have examined small components of system wide influences. Part of our goal for the research experiments carried out in the previous chapters was to attempt to provide a thorough characterization of how CB1Rs may impact cell signaling and influence neurodevelopment within a specific brain region, particularly the cerebellar cortex.

Previous cannabinoid work within the cerebellum has primarily consisted of CB1R immunostaining and Purkinje cell electrophysiological experiments of mature tissue, although the boundaries of what age constitutes developing tissue versus mature tissue are often unclear.

This is especially true with patch-clamp electrophysiology, where researchers have often opted towards recording from Purkinje cells as young as PND 8, which are smaller and ultimately more conducive to being voltage-clamped178. We have aimed to set more transparent boundaries regarding which ages constitute developing and mature tissue, although there is likely a continuous flux of protein expression throughout the time periods we have established.

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Developmentally restricted CB1R expression advances the concept that cannabinoid receptors have some importance on synaptic signaling during a developmental critical period. As our data presented throughout Chapters 2-4 shows, there is a system of CB1R regulation at multiple synapses throughout the developing cerebellar cortex, there are impacts on vesicle release in other neurotransmitter systems following CB1R activation, and there are clear effects

CB1R activation has on cerebellar-dependent behaviors. All of the observed effects provide an extensive, albeit hardly comprehensive, understanding of how the endocannabinoid system may influence synaptic function during a developmentally active time period.

CB1R localization and effects on synaptic transmission

In Chapter 2 we outline a thorough characterization of CB1R localization within the cerebellar cortex. This was achieved using immunohistochemistry as a visual guide, and was confirmed using a systematic step-by-step approach utilizing patch-clamp electrophysiology at each major neuron. These two approaches revealed functional presynaptic CB1R expression at many of these major synapses, excluding terminals onto Purkinje cells, which provided evidence that CB1R expression is developmentally transient in nature and is likely an important neurodevelopmental modulator.

Using immunohistochemistry we attempted to capture the localization and expression of

CB1Rs, in conjunction with known florescent markers, at multiple periods of development.

Images of mature (PND 30+) cerebellar cortex confirmed previous literature reporting extensive expression within the molecular layer and at basket cell -> Purkinje cell terminals (Fig. 2.1A). We also examined CB1R expression within the granule cell layer and saw its colocalization with

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glutamic acid decarboxylase (GAD) 65 & 67, a GABAergic cell marker, indicating it is likely expressed within granule cell layer glomeruli, masses comprised of mossy fiber and Golgi cell axons with granule cell dendrites. There appears to be a lack of literature addressing CB1R expression at mature granule cell synapses in cerebellar slice, so we naturally decided to determine whether a CB1R agonist (WIN 55,212-2) could alter synaptic signaling onto granule cells. What we found—a reduction in Golgi GABAergic vesicle release, but no change in mossy fiber glutamatergic vesicle release—would not have been surprising had we not determined WIN dramatically reduces mossy fiber vesicle release in immature, developing tissue.

Immunohistochemical findings in developing tissue display rapidly changing morphology coupled with dynamic CB1R expression throughout multiple layers of the cerebellar cortex. For example, in PND 4 tissue, expression patterns of CB1R appear more diffuse throughout all layers, especially the granule cell layer and surrounding undeveloped Purkinje cells (Fig. 2.3.A). This expression appears to become more spatially refined with the continual arrival of additional postmigratory granule cells and the arborization of Purkinje cell dendritic trees, with CB1Rs beginning to preferentially reside within the growing molecular layer by PND 9 (2.3.B). CB1R expression appears within granule cell layer glomeruli, where it colocalizes with vesicular glutamate transporter (VGlut) 1 & 2. We currently lack immunohistochemical staining in mature tissue containing both CB1R and VGlut 1 & 2 antibodies, but we could predict a lack of colocalization due to WIN’s lack of effect on mature mossy fiber afferents. Admittedly, an ideal immunohistochemical glomeruli comparison between developmental periods (PND 7 vs 32) would compare either GAD 65/67 or VGlut 1/2 at both ages. Regardless, the electrophysiological data ultimately provides a more accurate assessment of functional CB1R localization within the

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synapse. We should also consider our electrophysiological techniques would fail to detect either nonfunctional CB1Rs or CB1Rs that play a role beyond its typical mediation of synaptic transmission (such as CB1R-expressing proliferating granule cells lacking synapses145. Such CB1Rs could potentially explain heavy CB1R immunohistochemical expression within the developing molecular layer, despite a lack of functional CB1R actions on synapses terminating onto Purkinje cells.

The immunohistochemical data, coupled with electrophysiological data, project a cerebellar system rich in functional CB1R expression throughout development. The CB1R agonist

WIN 55,212-2 strongly reduces presynaptic vesicle release in a number of synapses within the cerebellar cortex during a third trimester equivalent rodent model; this effect is maintained in developed/adolescent tissue, but appears to exhibit shifted expression patterns. For example, mossy fiber terminals onto cerebellar granule cells appear to express CB1Rs in developing tissue, but this expression—functional expression, at least—dissipates once the cerebellum begins to display mature neuronal morphology and only post-migratory granule cells remain within the granule cell layer (PND 19+). Contrarily, Purkinje cells during early cerebellar development display no change in synaptic activity following application of the CB1R agonist WIN 55,212-2, but there is a striking shift in synaptic response shortly thereafter, where CB1R activation reduces vesicle release probability of both excitatory glutamatergic and inhibitory GABAergic synaptic release following WIN application (PND 16163; see Linden, 2000). To our knowledge, only terminals onto

Purkinje cells have been extensively studied in the past, including glutamatergic granule cell parallel fibers and climbing fibers, and GABAergic molecular layer stellate and basket cells. The one exception may be a study demonstrating Purkinje cell-produced endocannabinoids

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retrogradely activate molecular interneuron CB1Rs to reduce their firing rate166. Our data characterizes functional CB1R expression in a large proportion of neurons within the cerebellar cortex between two developmental periods.

In general, our electrophysiological data reveals effects of CB1R activation at every recorded cell type other than developing Purkinje cells, exemplifying both the importance of cannabinoid signaling developmentally and the transience of that signaling in maintaining appropriate neurotransmission. Determining CB1R agonists can reduce synaptic release at individual synapses provides us with an understanding of where CB1Rs are expressed and how they can influence monosynaptic neurotransmission, but equally important is determining whether this reduction in synaptic transmission can reduce downstream signaling.

Cerebellar network effects of CB1R activation

In Chapter 3 we were able to determine that CB1R activation reduced mossy fiber-evoked polysynaptic glutamate release onto targets within the molecular layer; namely, Purkinje cells and NMDA receptor-expressing migrating granule cells. This was presumably due to dampened granule cell excitation from CB1R actions on mossy fiber terminals rather than CB1R actions on parallel fibers. We can assume this because the CB1R agonist WIN had no effect on spontaneous or locally evoked glutamatergic vesicle release at the parallel fiber to Purkinje cell synapse.

However, the potential remains that parallel fibers express CB1Rs that act to suppress glutamate release onto migrating granule cells. We know at least some parallel fibers must express functional CB1Rs since we have established the CB1R agonist WIN reduces parallel fiber glutamate release onto Golgi cells, so we cannot rule out this possibility. Regardless,

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demonstrating that CB1R activation reduces mossy fiber-evoked glutamate release onto Purkinje and migrating granule cells indicates cannabinoids are able to modulate concentrations of neurodevelopmental regulatory molecules. Although intrinsically interesting, our rationale for examining polysynaptic mediatory properties of cannabinoids is based on the well-established role glutamate plays as a developmental signaling molecule42,62,264. Glutamate acts as a migratory guidance cue40,265 and is heavily involved in proper synaptogenesis between glutamatergic granule cell parallel fibers and Purkinje cell dendritic trees61,266. This is likely modified by the endocannabinoid system in vivo as an important synaptic regulator, but CB1R activation by exogenous cannabinoids, such as those contained within cannabis, have the potential to inappropriately modify synaptic signaling at a developmental period when tightly coordinated cues are essential.

Despite our focus, synaptically-regulated glutamatergic signaling only makes up a portion of the developmental milieu of signaling molecules. For example, granule cell migration rate is slowed, but not halted completely, following administration of saturating concentrations of

NMDA receptor blockers40,45, and isolated granule cells appear to exhibit internal cues that allow them to migrate independently50. The endocannabinoid system has also been implicated in regulating neurodevelopment in ways beyond synaptic control. As mentioned previously, intracellular mechanisms triggered through CB1R activation produce a plethora of effects, sometimes with opposite results. These include the facilitation of neuronal migration and axonal projections144,149,151,235, but also the repulsion of growth cones148. We have found an indirect method in which CB1R activation could potentially alter granule cell migration, but it is not a comprehensive measure of cannabinoid actions on developmental processes contained within

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the cerebellar cortex, and we should therefore not exclude other possible cannabinoid neurodevelopmental influences beyond ambient glutamate regulation.

Our demonstration that CB1R activation at the mossy fiber -> granule cell synapse is able to reduce synaptic transmission at the granule cell (parallel fiber) -> Purkinje cell synapse provides an ideal example of how reduced upstream CB1R activation can reduce overall synaptic signaling through the entire cerebellar circuit. Again, this is scientifically interesting in its own right, but more importantly has medical implications that could affect human health, considering cannabis also binds to these receptors. We attempt to address these concerns in the subsequent chapter.

Exogenous cannabinoid influence on cerebellar development

In Chapter 4 we report that rat pups exposed to the synthetic CB1R agonist WIN 55,212-2 during a specific developmental window indicative of human third trimester fetuses show deficiencies in navigating a cerebellar-dependent rotarod task during adolescence. We fail to link these deficiencies to an alteration in granule cell signaling (at PND 30-35), as we report no differences in granule cell synaptic input from mossy fibers or Golgi cells during baseline conditions or following WIN administration. This may suggest the motor deficiencies arise from alternations in a different cell type. Although we did not record the activity of climbing fibers onto Purkinje cells, previous literature has shown climbing fiber synapses to contain CB1Rs, and are able to reduce glutamate release into the synapse161,178. Climbing fibers are known to have additional Purkinje cell innervations during development before pruning back to a single climbing fiber per Purkinje cell in the mature synapse. It is possible that inappropriate CB1R activation could alter proper

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timing of this developmental action, as proper climbing fiber synaptic pruning relies on glutamate255,256, some of which is specific to parallel fibers267. If this were true, it could potentially explain the discrepancy between the rotarod task impairment of WIN-injected rat pups and a significant lack of differences in granule cell synaptic activity. Climbing fibers are known to send motor error signals to the cerebellum in attempt to correct them268, so rather than altered synaptic signaling at the level of granule cells, issues may arise at the climbing fiber->Purkinje synapse66, preventing proper motor error signals to correct improper motor issues It may be wise to pursue this further by determining basal climbing fiber activity following WIN exposure during development in the future.

Granule cell parallel fibers are a major source of glutamate for developmental processes, but there are additional sources beyond the scope of what we have experimentally tested for that may have intimate ties to glutamate regulation modified by endocannabinoids. CB1Rs coupled to Gq/11 G-proteins have been found on hippocampal astrocytes, and can activate phospholipase C and produce inositol triphosphate, which leads to mobilization of internal Ca2+ stores and stimulates glutamate release, evoking slow inward currents in CA1 pyramidal neurons117. This provides evidence of a non-synaptic glia-neuron interaction with involvement between endocannabinoids and glutamate, increased ambient glutamate levels could result in an opposite effect on migratory patterns if a similar mechanism exists during the granule cell migratory period within the cerebellar cortex. There has been reported CB1R expression on cerebellar astrocytes269, but our preliminary immunohistochemical images fail to show signs of

CB1R and GFAP (astrocyte marker) colocalization (Fig. 2.4), at least within the molecular layer and external granule layer. Additional experiments may be of interest to determine whether

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cerebellar astrocytes do truly express CB1Rs, optimally by recording intracellular Ca2+ levels from astrocytes in the presence of a CB1R agonist.

Although loosely connected, there are parallels we can make between the effects of synthetic cannabinoids on development in rodent models of third trimester pregnancy and the implications of pregnant women consuming cannabis or synthetic cannabinoids contained within

“spice”. We know that phytocannabinoids are likely able to activate the cannabinoid system within the cerebellar cortex at this age based on previous studies examining the dosage of THC in fetal tissue179,182,196,270, but there are limitations to the conclusions we can draw. For example, the synthetic cannabinoid WIN 55,212-2 has far greater affinity to CB1Rs than the partial agonist

THC131, so effects seen using the synthetic are probably artificially exacerbated compared to those likely occurring with the consumption of phytocannabinoids in vivo. However, due to the nearly universal illegality of cannabis, there is a market for the synthesis and consumption of synthetic cannabinoids, often referred to as “spice,” to circumvent drug prohibition laws271. This encourages individuals to ingest cannabinoids with much higher CB1R affinities than WIN, and far greater than THC122, which can result in overwhelmingly adverse physical and psychological reactions271,272. Although not as common as consuming cannabis, we should still consider these alternatives when factoring the developmental effects we see on cerebellar-dependent behavior.

Future directions

As mentioned prior, there are many additional experiments that remain to fully characterize how the endocannabinoid system mediates neurodevelopment within the cerebellar cortex, and how this correlates to normal function in vivo. For instance, it would be worth exploring the extent of

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endocannabinoid production and endogenous signal dampening within developing synapses, and how cannabinoid receptor activation influences synaptic plasticity. There are also a number of additional receptor types that endocannabinoids are thought to activate, potentially within the cerebellum, including GPR55100 and TRPV198,99. This also includes the potential expression and function of CB2Rs in the cerebellar cortex. CB2Rs have been found to promote stem cell/progenitor proliferation79,81, and are reportedly expressed on cerebellar microglia273. There is high immunoreactivity for CB2Rs within the cerebellar cortex, especially near what seems to be Bergman glial fibers, or near the Bergman glial fibers, although good antibody specificity for

CB2Rs is currently lacking. Developing a more comprehensive catalog of cannabinoid targets would further reveal endocannabinoid function in both developing and mature cerebellar cortex.

Finally, pinpointing an altered cerebellar cellular phenotype to account for the deficits seen in accelerating rotarod proficiency would provide an essential link to aid in understanding how CB1R signaling during development proves maladaptive. A first step may be to manipulate cerebellar granule cells specifically in an in vivo model, which would be a step toward isolating cerebellar dysfunction from other brain regions. This could also prove enlightening in understanding how developmental network functions coordinate migration, dendritic proliferation, and synaptogenesis within cerebellar circuitry.

As mentioned in Chapter 1, the cerebellum is quickly gaining recognition as a brain region involved in non-motor behaviors, and may be intimately involved in the development of neurodevelopmental disorders such as autism spectrum disorder (AUD)15,209,210,212,214. Aberrant neuronal migration appears to manifest in AUD and other neurodevelopmental disorders, and individuals with AUD display exhibit signs of altered migration274. This includes what appears to

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be disrupted cerebellar morphology in individuals with AUD275. This increases the relevancy of our research considering the intimate role glutamate plays in many facets of development such as neuronal migration, synaptogenesis, and dendritic pruning, coupled with our observations the developmental CB1R activation reduces downstream glutamate release.

Concluding remarks

We have developed a fairly comprehensive picture of where CB1Rs are expressed within the developing cerebellar cortex, how they control synaptic signaling through the circuit onto various synaptic targets, and have characterized both cellular and behavioral consequences following synthetic cannabinoid exposure during this period. Previous work has shown modified glutamate levels affects various stages of cerebellar development on granule and Purkinje cells, which our data eludes to following CB1R activation. However, there are still additional studies that should be performed in order to strengthen our understanding of how endocannabinoids are normally involved in cerebellar development and how they can function to perturb standard developmental processes. These findings can also aid our understanding of developmental cannabinoid roles elsewhere. Despite entirely focusing on the cerebellum, other brain regions such as the hippocampus have also been found to contain high levels of CB1R expression and are important for neurodevelopment, so studying the impact exogenous cannabinoids have on both cellular and behavioral cerebellar processing may be applicable to potential effects seen throughout the rest of the brain.

We have mainly referred to the manipulation of the endocannabinoid system during neurodevelopment as primarily negative, although the ability to artificially modify the level of

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cerebellar synaptic signaling may prove to be beneficial in the future. For example, there are a number of neurodevelopmental diseases associated with either damage or improper cerebellar development, such as autism spectrum disorder, and manipulating cerebellar activity may help to assuage symptoms and development15. For example, if overactive synaptic release is leading to the formation of too many synaptic connections, dampening overall synaptic release could better ensure only the strongest synaptic connections were maintained, whereas weaker synaptic points could undergo normal synaptic pruning. Once we better understand what could be leading to the development of these disorders, the unique cannabinoid modulatory role could be exploited. However, our general lack of understanding in terms of exogenous cannabinoid

(e.g. phytocannabinoids in cannabis) exposure on third trimester fetuses is far more pressing, as a lack of impact awareness may enable pregnant mothers to consume cannabis-related products without regard to health of the developing fetus.

Overall, the culmination of data contained within this dissertation provides evidence of functional endogenous CB1R expression within the developing cerebellar cortex, a period equivalent to the cerebellar developmental phase in third trimester human fetuses. Activation of these receptors can significantly dampen both excitatory and inhibitory neurotransmitter release from a number of neuronal types within multiple cerebellar layers, which can lead to a reduction in downstream polysynaptic vesicle release. This includes the important neurodevelopmental signaling molecule glutamate, which, when manipulated, is known to significantly alter both granule cell migration and granule cell and Purkinje cell dendritic proliferation and synaptogenesis40,63,276. When exposing rat pups to the exogenous cannabinoid WIN 55,212-2, they show deficiencies in learning to navigate a cerebellar-dependent accelerating rotarod

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behavioral task, suggesting improper CB1R activation produces a developmental insult to the cerebellar cortex. Although we failed to detect differences in granule cell signaling following WIN exposure, we predict a number of cerebellar alterations which could produce a similar result.

These results support emerging evidence recognizing the pivotal role the endocannabinoid system performs in developmental processes and exemplifies the functional diversity of endocannabinoid function. CB1R activation has direct effects on synaptic modulation, but also clearly impacts other neurotransmitters at distant, polysynaptic sites. From a bigger perspective, these data may aid in the eventual production of guidelines used to outline safe cannabis usage for pregnant mothers, but may also detail a unique method in which dampening synaptic signaling could be beneficial, such as curtailing overstimulation of glutamate in nervous disorders such psychosis277,278 and autism spectrum disorder238.

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