Pleiotropic Signaling in the Endocannabinoid System: The role of the G protein γ3 subunit A THESIS SUBMITTED TO THE SCHOOL OF GRADUATE STUDIES of BLOOMSBURG UNIVERSITY OF PENNSYLVANIA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE PROGRAM IN MOLECULAR BIOLOGY DEPARTMENT OF BIOLOGY AND ALLIED HEALTH SCIENCE BY ALEX PASCULLE BLOOMSBURG, PENNSYLVANIA 2022 1 Abstract The Cannabinoid 1 (Cb1) receptor is increasingly recognized as being involved in numerous pathological and physiological processes. Classical Cb1 receptor signaling occurs at the pre-synaptic terminal, where it couples to Gαi/o proteins to inhibit adenylyl cyclase and mediate retrograde inhibition. It is now well established that Cb1 signaling is pleiotropic and occurs in many different cell types through the actions of different Gprotein alpha (α), beta (β) and gamma (γ) subunits. While Gβγ has been identified as having specific roles in this signal transduction process, the unique roles that individual Gγ subunits perform remains elusive. To explore these roles, we transiently overexpressed Cb1 and Gγ3 (Gng3) in a CHO-K1 cell line and measured cAMP accumulation following receptor activation. We hypothesized that overexpression of Gng3 would result in preferential coupling of Gγ3 into the Gαβγ heterotrimer and result in an altered response when compared to cells only expressing Cb1. However, lack of statistical significance and the high variation between trials lead us to reject this hypothesis. Similarly, we used this overexpression model to measure intracellular calcium levels in cells expressing Cb1 or Cb1+gng3. We found that stimulation of Cb1 with Anandamide had no effect on intracellular calcium in either group. Next, we used a CRISPR-Cas9 gene targeting approach against Gg3 in developing Danio rerio (zebrafish) embryos. Our approach at targeting the gng3 allele was successful, with target efficiency of up to 95% in the F0 progeny. Finally, we utilized a series of behavioral assays to measure Visual and Acoustic startle responses in wild type Zebrafish. Herein, we report on these experiments and how additional troubleshooting of these assays are needed before any claims can be made on the mutant phenotype. 2 Acknowledgements Words cannot express my gratitude to my Major Professor, Dr. Schwindinger, for his guidance and patience throughout this endeavor. This work would also not of been possible without the help of my committee members—Dr. Klingerman and Dr. Coleman, and the rest of the faculty in the Bloomsburg University Department of Allied Health Sciences. Furthermore, I would like to thank my friends and colleagues in the M.S. program for their help with editing, researching, discussing and moral support. Thanks should also go to Dr. Hranitz and Thomas O’Rourke for their help with the statistics, and to Ayushi Umrigar and Eric Moeller for helping with my experiments. Lastly, I would like to extend a special thanks to my family, especially my mom and sisters for always encouraging me to be my best. This project has given me a new appreciation for science and all things G-proteins. 4 Table of Contents ABSTRACT………………………………………………………………………………2 THESIS APPROVAL SIGNATURE PAGE………………………………………… 3 ACKNOWLEDGMENTS…………………………………………………………….…4 LIST OF TABLES………………………………………………………………………6 LIST OF FIGURES……………………………………………………………………7 LIST OF APPENDICES………………………………………………………………8 INTRODUCTION………………………………….……………………………………9 1. ENDOCANNABINOID SYSTEM………………………………………………….….……9 1.1 COMPONENTS OF THE ENDOCANNABINOID SYSTEM……………………9 1.2 THE BIOLOGICAL ROLE OF THE ENDOCANNABINOID SYSTEM…...…10 1.2.1 ECB IN THE NERVOUS SYSTEM……………………………….…11 1.2.2 THE ECB IN THE CARDIOVASCULAR SYSTEM…………….…12 1.3 THE ENDOCANNABINOID SYSTEM IN ZEBRAFISH……………….………13 2. G PROTEIN COULES RECEPTORS AND HETEROTRIMERIC G-PROTEINS……17 2.1 SYNTHESIS AND MODIFICATIONS OF G PROTEINS…………...…………18 2.1.1 Ga………………………………………………………………………18 2.1.2 Gb AND Gγ……………………………………………………………18 2.2 SIGNALING AND REGULATION………………………………………………20 3. CANNABINOID RECEPTOR 1…………………………………………………...………21 3.1 BIOLOGY AND DISTRIBUTION OF CB1……………………………...………21 3.2 SIGNALING AND G-PROTEIN COUPLING……………………………...……22 4. THE Gγ SUBUNIT………………………………………………………………….………24 4.1 BIOLOGY OF Gγ…………………………………………………………….……24 4.2 SPECIFICITY OF Gγ………………………………………………………..….…24 4.3 THE Gg3 SUBUNIT ………………………………………………………………26 METHODS …………………………………………………………………………29 RESULTS………………………………………...…………………………………44 DISCUSSION………………………………………………...…………………………50 REFERENCES………………………………………………….………………………84 5 LIST OF TABLES TABLE 1: NON-EXHAUSTIVE TABLE OF G-PROTEIN EFFECTOR PATHWAYS…98 TABLE 2: NON-EXHAUSTIVE LIST OF CB1 PLEIOTROPIC SIGNALING……,,,…100 TABLE 3: PCR PRIMERS……….………..………..………..………..………..………104 TABLE 4: AVERAGE DISTANCES TRAVELLED DURING STARTLE………..……112 TABLE 5: SURVIVAL RATES FOR INJECTION TRIAL 0………..………..………..117 TABLE 6: SURVIVAL RATES FOR INJECTION TRIAL 1………..………..……..….118 TABLE 7: SURVIVAL RATES FOR INJECTION TRIAL 2………..……..………..…119 6 LIST OF FIGURES FIGURE 1: THE ENDOCANNABINOID SYSTEM…………………...……………97 FIGURE 2: ACTIVATION OF A GPCR…………………………………………..…99 FIGURE 3: MAP OF PCDNA3.1 RESTRICTION SITES AND INSERTS………101 FIGURE 4: MAP OF PCDNA3.0 RESTRICTION SITES AND INSERTS………102 FIGURE 5: SCHEMATIC OF CRISPR/CAS9 TARGETING GNG3………….…103 FIGURE 6: EMBRYOS LINED UP BEFORE MICROINJECTION……….……105 FIGURE 7: PCR PRODUCTS OF CDNA ISOLATES…………..………..….……106 FIGURE 8: RESTRICTION ENZYME DIGEST OF PLASMIDS…………..……107 FIGURE 9: CAMP-GLO STANDARD CURVE………..………..………..……..…108 FIGURE 10: FURA-2AM CALCIUM TRACINGS………..………..………...……109 FIGURE 11: AVERAGE DISTANCE TRAVELED IN VMR ASSAY………..…..110 FIGURE 12: DISTANCE TRAVELLED IN AUDITORY STARTLE ASSAY…..111 FIGURE 13: HISTOGRAM OF STARTLE LATENCY………..………..………..113 FIGURE 14: PCR PRODUCTS OF CRISPANTS………..………..………..……...114 FIGURE 15: INDEL PLOT FROM CRISPANTS………..……..………..….……..115 FIGURE 16: CHROMOGRAM AND PHOTO OF CRISPANT………..…...…….116 7 LIST OF APPENDICIES APPENDIX 1: IACUC FORMS……..………..………..………..………..………..120 APPENDIX 2: SUPPLEMENTAL FIGURES………..………..………..………..133 8 1. INTRODUCTION 1.1. Components of the Endocannabinoid system The Endogenous Cannabinoid System is composed of the Cannabinoid receptors (Cb1 and Cb2), their endogenous ligands (endocannabinoids), and the enzymes responsible for the synthesis and degradation of the endocannabinoids. Narachidonoylethanolamide (AEA) and 2-arachidonylglycerol (2-AG) are the two principal ligands. Unlike classical neurotransmitters that are stored in secretory vesicles, the lipophilic nature of the two main endocannabinoids allows for simple diffusion across the lipid bilayer. Alternatively, endocannabinoids may be transported into or out of the cell through the Endocannabinoid Membrane Transporter (Nicolussi et al., 2015, Fowler 2013) (Figure 1). N-Arachidonoyl-ethanolamine, more commonly referred to as ‘Anandamide’, contains an ethanolamine conjugated to an eicosanoid derivative. Biosynthesis of Anandamide (AEA) occurs in a Ca2+ dependent manner by the integral membrane protein N-Acyl phosphatidylethanolamine Phospholipase D (NAPE-PLD). AEA is broken down by an integral membrane protein, Fatty Acid Amid Hydrolase (FAAH) (Cascio and Marini 2015, Wiley et al., 2018). Like AEA, 2-AG is also synthesized in a Ca2+ dependent manner by a membrane protein that sits within the inner membrane of the lipid bilayer. In the case of 2-AG, Diacyl Glycerol Lipase (DAGL) catalyzes the cleavage of 2AG from a diacyl glycerol molecule in the lipid bilayer (hill and tasker 2012). Despite sharing similar biochemical properties, key differences in their biosynthesis, degradation, 9 and receptor affinity allow the two ligands to perform selective or pleiotropic functions. AEA has been proposed to represent a tonic signal, continuously regulating neurotransmitter release, while 2-AG is responsible for a phasic signal that is required for synaptic plasticity (Kilaru and Chapman, 2020). Advancements made in fields such as biochemistry (Ropke et al., 2021), lipid signaling (Zamberletti et al., 2017), and pharmacology (Kaczocha and Dahmane, 2021) have allowed for an expanded definition of what is considered part of the endocannabinoid system. For instance, the identification of two other putative receptors with partial affinity to endocannabinoids—the orphan GPCR 55 (GPR55) (Ryberg et al., 2007) and the transient vanilloid type-1 (Trpv-1) channel (Muller et al., 2018; 2021) represent another ‘layer’ of signaling pleiotropy and further highlight ambiguities in what we currently know about this system. While the Endocannabinoid system encompasses numerous components, the scope of this project is limited to the Cb1 receptor and its activation by Anandamide. 1.2. The Biological role of the Endocannabinoid system Extracts from the plant Cannabis sativa has been used for centuries to treat conditions ranging from chronic pain to epilepsy. First credited with introducing cannabinoids into Western medicine was Irish physician William O’Shaughnessy (W. B. O’Shaughnessy, 1843). However, it was not until the cloning of the Cannabinoid 1 receptor (Cb1) by researchers at the National Institutes of Health (NIH) (Matsuda et al., 1990) that the mechanisms of how these effects are attained were first explored. Since then, a large body of experimental literature has been published speculating the biological role of the cannabinoid system (see review Skaper and Marzo et al., 2012). Here I present just a few 10 instances showing the ubiquity of the endocannabinoid system and its role in many biological systems. 1.2.1. The Ecb in the Nervous System The Cb1 receptor is the most abundantly expressed receptor in the central nervous system (CNS) (Hu, S. et al., 2015). Despite its widespread distribution, teasing out the physiological role for the Cb1 receptor in humans has been a daunting task. One apparent function of the Cb1 receptor is to attenuate analgesia. Pernia-Andrade et al., used paired pulse and extracellular electrophysiological recording experiments to show that the synthetic Cb1 receptor agonist WIN activates receptors on inhibitory dorsal horn interneurons and subsequently reduces the release of GABA and Glycine onto nociceptive C fibers (Pernia-andrade et al., 2010). Preclinical models using Cb1 receptor agonists in model organisms have also been able to exploit the anti-nociceptive properties of Cb1 through peripheral and central injections of Cb1 receptor agonists. Moreover, these effects were counteracted using selective Cb1 antagonists (Racz et al., 2015; J. Lotsch, et al., 2017). Activation of the cannabinoid receptors has been proposed to alleviate the symptoms and progression of many different neurodegenerative diseases. Early support for a role of the endocannabinoid system in neurodegenerative pathologies came about when alterations of endocannabinoid signaling components were observed in the cerebrospinal fluid (Di Filippo et al., 2008), blood (Jean-Gilles et al., 2009) and neural tissue (Centonze et al., 2007), in both human and animal models of disease. These alterations in the expression pattern and distribution of Cb1 in different models of disease 11 suggest a role for the endocannabinoid system in neuropathologies (See review Cristino et al., 2020). 1.2.2. The ECB in the Cardiovascular System At the molecular level, Cb1 is expressed by endothelial cells in the tunica intima, vascular smooth muscle cells (VSMC) in the tunica media, and cardiomyocytes in the myocardium. While the biological function of Cb1 in these tissues remain elusive, Cb1 is significantly upregulated in response to cardiac ischemia and tissue damage. When activated under these conditions, the majority of Cb1 signaling occurs from activation of the sympathetic, and inhibition of the parasympathetic nervous system (Sara-lena Puhl 2020). Like many other receptors, Cb1 also activates non-classical G protein pathways. For instance, In vitro application of Cb1 agonists and antagonists to rat (Domenicalli et al., 2005) and human (Stanley et al., 2016) mesenteric artery cause vasorelaxation through the activation of Endothelium derived Nitric oxide synthase (ENOS) from endothelial cells. In this scenario, AEA activation of Cb1 on endothelial cells causes the G-protein dependent activation of the MAPK cascade (J liu, 2000)—ultimately leading to activation of ENOS and the release of Nitric Oxide (NO) which will cause local vasodilatory effects on the VSMC surrounding the vessel (Stanley, 2016). Similarly in cardiomyocytes, Cb1 receptor activation causes an overall negative inotropic effect and thereby decreasing contractility (S. Batkai, et al.,2004). Cb1 activation on peripheral nerve terminals of post ganglionic sympathetic neurons leads to a decrease in the amount of Norepinephrine (NE) released by Sympathetic neurons and thereby decreases the “sympathetic tone” (S. Batkai, et al.,2004). On the 12 contrary, in the CNS, activation of Cb1 causes an increase in sympathetic tone. When AEA is administered systemically, an overall increase in sympathetic tone occurs. The opposing effects of AEA acting systemically as a positive inotrope and locally as a negative inotrope can be partially reconciled when considering a protective role for the Endocannabinoid system in cardiovascular pathology. While central injection of AEA results in an overall increase in sympathetic tone (S. Batkai, et al.,2004), local release of AEA causes an overall decrease in sympathetic tone serving as a protective mechanism against ischemia and reperfusion injuries (Maeda et al., 2009). While the function of the endocannabinoid system in the cardiovascular system is outside the scope of this thesis, it demonstrates the ubiquity of Cb1 signaling in multiple systems and highlights how a strong understanding of the molecular events governing these responses can create endless possibilities in treating a range of pathologies. 1.3. The Endocannabinoid system in zebrafish Zebrafish have been a robust tool for researchers investigating various aspects of biology and pharmacology in vertebrates. This is in part because of the high genetic homology (~70%) between zebrafish and humans (Howe et al., 2013) and the presence of approximately 84% of orthologous genes known to cause disease in humans (Grunwalk et al., 2002). Sequencing of the Cb1 receptor in zebrafish revealed a 69% nucleotide and 73.6% amino acid sequence homology when compared to Cb1 in humans (Lam CS., et al.,2006). Zebrafish also contain orthologs to the human genes encoding the various other components of the endocannabinoid system including the enzymes NAPE-PLD, FAAH and DAGL (Migliarini B and Carnevali o, 2006). Another advantage in using zebrafish as biological models is the presence of a clear chorion and easy methods of genetic 13 manipulation that make visualizing and characterizing the developing larvae relatively easy and amenable to high throughput assaying when compared to other model organisms (Choi et al., 2021). Following a similar expression profile as their mammalian counterparts, zebrafish begin expressing Cb1 in the preoptic center of the hypothalamus at the 3-somite stage of development (24 hpf) (CS lam 2005). The expression pattern of Cb1 in the developing zebrafish dorsal telencephalon coincides with the development of inhibitory GABAergic neurons. Interestingly, Cb1 was preferentially expressed in a subset of neurons in the locus coeruleus that give rise to the Vth cranial nerve (Trigeminal nerve). One interpretation of this finding could be that Cb1 regulates the release of GABA, which modulates the inhibitory activity in the ventral striatum (Watson et al.,2006) and subsequent release of dopamine onto dopaminergic neurons in the substantia nigra. Moreover, intense signals in the diencephalic posterior tuberculum (homologous to the mammalian mid-brain dopamine system) and the medial zone of the dorsal telencephalon suggests that the Cb1 may be involved in reward-related behaviors, hippocampal and memory formation, and other cognitive processes (CS lam 2005). This interpretation is only speculative and may be misleading considering the researchers were only concerned with the temporal and spatial patterns of expression of Cb1. Nevertheless, it provides empirical support for the involvement of Cb1 in the development and maintenance of various structures and processes in the CNS. Elucidating the intricate and ubiquitous expression patterns of Cb1 throughout development has proven to be a daunting task. Pharmacological perturbation and gene targeting strategies have been of tremendous value for researchers interested in ascribing 14 biological function to the endocannabinoid system. An excellent example of this occurred in 2008 when Watson et al., demonstrated that pharmacological inhibition of the Cb1 receptor in developing zebrafish led do defects in axon pathfinding and fasciculation in the striatum (Watson et al.,2008). A similar yet more exaggerated phenotype was observed in embryos injected with antisense morpholinos (MO) to the Cb1 receptor. 100% of the developing morphants exhibited significant disorganization and loss of fasciculation in tracts of the medial longitudinal fasciculi (MLF) (Watson et al.,2008). This led researchers to conclude that axonal elongation, pathfinding, and fasciculation is mediated, at least in part, by the Cb1 receptor. There have been relatively few zebrafish studies aimed at functionally characterizing the Cb1 receptor in terms of its biological role in the endocannabinoid system. While this may be attributed to the growing interest in studying exogenously derived cannabinoids like CBD and THC to treat diseases, it highlights a large gap in our knowledge regarding the basic phenotypes involved in endocannabinoid signaling of lower vertebrates. Nevertheless, activation of Cb1 in zebrafish has been shown to produce conflicting phenotypes. For instance, Connors et al., (2014) reported an apparent anxiolytic-like response in adult zebrafish treated with the synthetic Cb1 receptor agonist WIN55212-2. This comes in stark contrast to other studies where Cb1 activation caused anxiogenic-like responses (Stewart & Kalueff 2014, Ruhl et al.,2016). These later studies agree with preliminary data generated from the Klingerman lab which suggests activation of the Cb1 receptor in adult zebrafish increases locomotion and anxiety related behaviors (in press, Schaffer and Moeller 2020) Collectively, these results may suggest the emergence of distinct, yet conserved functions mediated by the Cb1 receptor. 15 Zebrafish possess a large repertoire of innate behavioral responses that are “hardwired” into their brain. The sensory-motor circuits responsible for eliciting these responses must provide the organism with the ability to detect and avoid predators and guide them into a safe and nourishing environment (Neuhauss, 2003;Burgess and Granto, 2007;Vaz et al.,2019). Similar to the pupillary response that occurs in mammals exposed to a photic stimulus, zebrafish have also been shown to display varying optomotor responses. Previous studies have described a distinct series of behaviors that occur in response to abrupt changes in illumination and are thought to facilitate an escape from overhead predators (Easter and Nicola, 1997; Orger and Baier, 2005; Lutchenburg et al., 2019). These responses are collectively referred to as the Visual Motor Response (VMR) and occurs when zebrafish are exposed to sudden dark or flashes of light. The VMR is often initiated several hundred milliseconds (ms) after presentation of light flashes and results in higher levels of locomotor activity in zebrafish (when compared to basal levels) (Burgess and Granto, 2007). Several studies have used the VMR in zebrafish as a behavioral paradigm to ascribe function to various aspects of zebrafish biology. Recent discoveries have identified the trigeminal nerve as having crucial functions in the VMR of zebrafish (koshashi 2012 and koide 2018). Here, we attempted to use the VMR assay to characterize the phenotypes of zebrafish mutants and to further understand the role of the endocannabinoid system in primitive responses. As discussed later, the mutant zebrafish generated in this study did not develop to 7dpf when the assay was planned to occur and thus prevented us from generating VMR data on mutants. Like the VMR displayed early in the development of zebrafish larvae, the auditory startle response is an innate response that develops around 5dpf (Zeddies and Fay, 2005). 16 This response is initiated by a high-intensity acoustic stimulus that activates a large group of reticulospinal neurons called Mauthner cells (M-cells). After activated, M-cells synchronously synapse to contralateral spinal motor neurons causing a characteristic “Cbend”. The C-bend acts to propel fish away from the stimulus or perceived threat (Eaton et al., 2001). Considering that the Cb1 receptor is often involved in the inhibition of retrograde neurons in the CNS, we used this acoustic startle paradigm to determine weather Cb1 was involved in enhancing or limiting this response in juvenile zebrafish. 2. G Protein Coupled Receptors and Heterotrimeric G-Proteins Containing over 800 members encoded in the human genome, G-Protein Coupled receptors (GPCRs) are the most abundant superfamily of membrane protein receptors in mammals and a major target for pharmacological agents. GPCR’s transduce extracellular signals like photons or neurotransmitters into an intracellular signal that is amplified through a G-protein signaling cascade. GPCRs are classified into five families based on sequence homology and functional similarity. The vast majority of these proteins belong to family A or Rhodopsin-like receptors (Syrovatkina et al., 2016). All GPCR’s share similar structural features and are characterized by an extracellular N-terminus, and intracellular C-terminus and seven trans-membrane spanning α-helices. When the receptor is in its inactive state, the intracellular domain is associated with the heterotrimeric G protein composed of alpha (α), beta (β) and gamma (γ) subunits. While the α subunit is GDP-bound, the β and γ subunits are tightly associated with each other. The receptor is then activated by agonist binding which catalyzes the exchange of GTP for GDP followed by the disassociation of the Gα-GTP bound subunit from the βγ dimer. Both Gα and Gβγ will activate signaling cascades. The signaling is terminated 17 following the hydrolysis of GTP to GDP catalyzed by the biophysical GTPase properties of the Gα subunit (Weis, W. et al.,2018, NCBI; Figure 2). 2.1. Synthesis and modifications of G proteins 2.1.1. Gα Humans contain 16 genes that code for Ga subunits, which are classified into four families based on sequence and functional homology. These families— Gαs Gαi/o, Gαq and Gα12/13 = are broadly responsible for stimulating Adenylyl Cyclase (AC), Inhibiting AC, activating PLC and activating RhoGEFs, respectively (Hollmann et al., 2005). Considering that these responses are not solely mediated by the identity of the Gα subunit, these responses have also been used to characterize the canonical response of the GPCR. There are two different types of modifications that Gα can undergo— myristoylation and palmitoylation. These lipid modifications allow the Gα protein to remain associated with the inner leaflet of the plasma membrane (Mumby et al., 1990). In addition to the lipid modification incurred by Gα, yet another important factor in membrane anchorage is found to be encoded right in the amino acid (AA) sequence— the polybasic motif. In many of the Gα subunits, a stretch of around 10 positively charged AA’s are found grouped together in its Amino terminal and is believed to facilitate interactions with the negative membrane surfaces (Kosloff 2002). 2.1.2. Gβ and Gγ There are at least five beta subunits that are encoded for by different genes giving rise to the Gβ 1-5 subunits. Gβ 1-4 share greater than 80% amino acid homology compared to only 50% with Gβ5 (Smrcka, 2008). Several Gβ subunits also contain 18 polybasic motifs located on the amino terminus. Polybasic motifs in Gβ are a conserved sequence of positively charged amino acids that are also believed to facilitate membrane anchoring through its interactions with the acidic phospholipid membrane (Murray et al., 2001). Nascent Gβ and Gγ subunits dimerize in the cytoplasm before being recruited to the ER, where these modifications occur post translationally (Murray et al., 2001). Several putative molecular chaperones like Chaperonin Containing Tailless-complex polypeptide 1 (CCT) (Lukov et al.,2006) and the ER resident Dopamine Receptor interacting Protein 78 (DRiP) 78 (Dupre,2007) have been identified that coordinate the processing and shuttling of G-proteins within the cell. These proteins are thought to contribute to the assembly of a specific Gβxγx dimer (Marrari et al., 2007). Assembly of a preferential Gαβγ heterotrimer is one of the many factors thought to contribute to signaling pleiotropy. The lipid modifications that occur to the γ subunit are done by specific transferases known as farnesyl and geranylgeranyl transferases (Marrari et al., 2007, Hynes et al., 2004). Prenylation is a unique modification that occurs in CaaX box proteins. The CaaX box is a combination of the last 4 amino acids found at the C terminal of the protein. The C stands for cysteine, meaning that a cysteine residue must be the fourth-to-last amino acid (Hynes et al., 2004). This is the amino acid where the prenyl moiety, either farnesyl or gerynylgerynyl, will be added. The two “a” represent the presence of some aliphatic amino acid and the amino acid in position X is what determines which of the two prenyl groups (Farnesyl and Geranylgeranyl) will be added (Hynes et al., 2004). 19 For the addition of a farnesyl group, the amino acid in the X position needs to be either an alanine, serine, or methionine. Gγ 1, 9 and 11 all have a serine residue in the C position and are therefore farnesylated. For the addition of a geranylgeranyl group, the amino acid in position X must be a leucine. The rest of the Gγ proteins all contain a leucine in the X position and are thus geranylgeranylated. In contrast to the lipid moieties added onto the Gα subunit, processing of Gγ requires post-prenylation modifications before it can properly assemble. The first post-prenylation modification is the proteolytic removal of the aaX Amino acids, so that the prenylated cysteine residue is the first Amino acid at the C terminus. Following aaX proteolysis comes methylation of the Cterminal carboxyl group. Carboxymethylation is important because it contributes to the hydrophobicity of the C terminus (Evanko et al., 2000) thereby facilitating anchorage to the membrane. 2.2. Signaling pathways and Regulation By convention, GPCRs have been characterized by the identity of the Gα subunit by which it preferentially couples to. This is because of data that has accumulated over the years identifying well defined or Canonical signaling pathways mediated by Gα (Table 1). The family Gαs was named as it is responsible stimulating Adenylyl cyclase (AC), which converts ATP to cAMP leading to an increase in intracellular cAMP. In contrast, Gαi/o was named after its inhibitory effect on AC leading to an increase in intracellular cAMP. The family Gαq is responsible for activating phospholipase C beta (PLCbeta) which hydrolyzes Phosphatidylinositol 4, 5-biphosphase (PIP2) into diacylglycerol (DAG) and inositol 1, 4, 5-triphosphate (IP3) causing the subsequent opening of IP3 channels on the ER and release stored Ca2+ (Syrovatkina et al., 2016). 20 Throughout this thesis, we will refer to these responses generally as Gs Gi/o and Gq to eliminate confusion when discussing the role of Gγ in G protein signaling. In addition to signaling pathways mediated through Gα proteins, Gbg dimers also initiate signaling through a wealth of well-defined effectors (Syrovatkina et al., 2016). The existence of these non-canonical signaling pathways has made characterizing GPCR response difficult and are often overlooked by researchers who are strictly defining GPCR’s as signaling through their Gα subunit. Table 1 shows a non-exhaustive table of well-defined effectors for Gα and Gβ/γ and their physiological relevance. 3. Cannabinoid Receptor 1 3.1. Biology and Distribution of the Cb1 receptor Cb1 is encoded by the intronless CNR1 gene found on chromosome 6 locus q14-q15 andconsists of a 472 amino acid sequence sharing 97-99% sequence homology with rat and mouse. Multiple Isoforms coming from the 5’-UTR have been identified. CNR1 also contains three noncoding exons allowing for the alternative splicing variants Cb1A, Cb1B, Cb1C, and Cb1D. These isoforms are a result of intraexonal splicing events of CNR1 and contain a variable 5’untranslated regions (González-Mariscal, I. et al., 2016). The presence of these splice isoforms is only one example of the different post transcriptional mechanisms responsible for signaling pleiotropy. A high concentration of Cb1 has been observed in the presynaptic terminals of neurons found within the nervous system, where it functions as a retrograde signaling mediator by inhibiting the influx of Ca2+ into the presynaptic cell—thus hindering the release of neurotransmitters from that cell (Katona, I. et al.,1999 ,Hu, S. et al., 2015). 21 Ultimately, this leads to long- and short-term effects on synaptic plasticity where it often modulates multiple neuronal circuits and functions as a homeostatic regulator of neuronal excitability (wright et al., 2017). Cb1 is most abundantly expressed in the olfactory bulb, hippocampus, basal ganglia and cerebellum. Moderate levels of Cb1 expression have been reported in the cerebral cortex and dorsal horns of the spinal cord. In contrast, lower expression has been reported in the ventral horn and thalamus (Veress, G. et al., 2013). In the Peripheral Nervous System (PNS) Cb1 is primarily present in the sympathetic nerve terminals, the trigeminal ganglion, Dorsal root ganglion, and in the dermic nerve roots (Clapper, J. et al.,2010) where it is thought to regulate nociception from afferent pathways. 3.2. Signaling Pathways and G-protein coupling The Cb1 receptor is a member of the rhodopsin family of G-Protein coupled receptors. It was initially proposed by Howlett and Fleming in 1984 that Cb1 exerts its biological effects through the preferential coupling to Gαi/o proteins. Support for this hypothesis came about when Howlett showed that cAMP production was inhibited upon D9-THC treatment in Cb1 expressing neuroblastoma cells and blocked when treated with pertussis toxin (PTX) (Howlett and Flemming, 1984). Moreover, [35S]GTPyS binding assays on Cb1 demonstrate high affinity to Gαi proteins (Priestley R, et al., 1998). Cb1mediated retrograde inhibition of presynaptic neurons occurs through both the downstream inhibition of cAMP/PKA via Gαi/o , and activation of G-protein inwardlyrectifying K+ channels (GIRKs) and inhibition of N-type, P/Q type Voltage gated calcium channels via Gβ/γ. Presynaptic calcium inhibition and the efflux of K+ through 22 GIRKS limits the excitatory or inhibitory response on the post synaptic neuron by hyperpolarizing the presynaptic membrane (Sudhof and Starke, 2004). More recently, the crystal structure of a signaling cannabinoid receptor 1 protein complexed with a Gαi protein has recently been solved (Krishna, K. et al., 2019). Indeed, support from these studies are what form the basis of this canonical view of Cb1 signaling. However, the canonical view of GPCR signaling fails to explain the differential signaling patterns in the Cb1 receptor. The Cb1 receptor can activate different signaling pathways depending on the receptor conformation induced by the activating ligand, a phenomenon known as functional selectivity or biased agonism. For example, Lauckner et al. compared intracellular Ca2+ response in HEK-293 cells stably transfected with Cb1 after treatment with different Cb1 agonists. Upon treatment of transfected HEK-293 cells with the synthetic agonist WIN55,212-2 (WIN), a transient influx of Ca2+ was recorded. This response was still present after cotreatment with Gαi sensitive PTX (Lauckner, J. 2005). The response was blocked when cells were treated with Phospholipase C inhibitors, suggesting that the response may also be mediated by Gq Proteins. Offering more compelling evidence for functional selectivity, a study conducted by Diez alarcia et al. in 2016 revealed that Cb1 can couple to the classic inhibitory Gαi/o proteins, in addition to different Gα subunits like Gαz, Gαq/11 and Gαq12/13 in a ligand specific manner. Using a combination of GTP γ biding assays and antibodies to specific Gα subtypes, it was determined that activation of the receptor by different ligands can significantly alter its response (Diez Alarcia R. et al.,2016). By using specific antibodies targeted to different inhibitory Gα proteins, Diez-Alarcia demonstrated the variability in 23 Gα protein coupling in response to the activation by a single ligand. These results imply that Cb1 is capable of coupling to different G protein subunits in a way that cannot solely be explained by functional selectivity or cellular context. Table 2 shows a non-exhaustive list of the multiple levels of signaling pleiotropy in the Cb1 receptor. 4. The Gγ subunit 4.1. Biology of the Gγ Subunit Assembly of the G-α β γ heterotrimer is a key step in the G-protein signaling cascade. With 16 α subunits, 5 β subunits, and 12 γ subunits, there are 960 different combinations of possible Gαβγ heterotrimers. Considering that many of these associations are physically possible, (Richardson and Robishaw 1999), the presence of preferential Gαβγ combinations that occur in specific cellular contexts offers support for a functional role for the γ subunit in eliciting a physiological response. In contrast to their β subunit counterparts and arguing against an eponymous “Gβγ dimer,” the γ subunits exhibit more variation in amino acid homology and tissue specific distribution suggesting an emergence of distinct functions. The current repertoire of 12 g subunits (coded for individual genes) diverged from 5 ancestral subunits to form the following classes—Class I: Gγ7 and Gγ12; Class II: Gγ2, Gγ3, Gγ4, and Gγ8; Class III: Gγ5 and Gγ10; Class IV: Gγ1, Gγ9 and Gγ11; Class V: Gγ13 (Kahn 2013, Syrovatkina et al.,2016). 4.2. Specificity of the g subunit Sensory cells offer a great example of how signaling specificity can be achieved through the actions of individual Gγ subunits. This was demonstrated early on when Peng et al., showed an absolute requirement for Gαt1, β 1 and γ 1 for night vision in retinal rod 24 cells and G αt2β3γ8 for color vision in retinal cone cells (Kolesnikov et al., 2011; Peng et al., 1992). It was later determined through gene knockout studies that loss of Gγ1 effects membrane localization of the Heterotrimeric G-protein complex and subsequent degradation of Gαt1 (Lobanova et al., 2008). Similarly, Kerr et al., demonstrated a novel role for Gαolfβ1γ13 in olfactory neurons that are responsible for eliciting olfaction. Whereas transcription is often the main driver for the assembly of preferential heterotrimeric complexes in sensory and other specialized cells, it is unlikely the case in many other cell types that express an array of G-proteins and yet still have specialized functions. Compelling evidence for a post-translational mechanism governing the assembly of distinct Gαβγ heterotrimers first came about in 2003 when researchers at the Weis center for research used reverse genetic approaches to generate Gng7-/- knockout mice and show a novel role for γ7 in D1 dopamine and A2a Adenosine receptor signaling in the rat striatum (Schwindinger 2003, 2010). Quantitative immunoblot analysis revealed that these mice showed a stoichiometric reduction of Gαolf and Gβ2 proteins in the cytosol and membrane extracts, while the levels of their mRNA transcripts remained unchanged. These mice also exhibited distinct phenotypes. For instance, Gng7-/- exhibit a reduction in AC activity and a particular behavioral phenotype that is consistent with complete or partial loss of reward and locomotive related behaviors. Researchers speculated that because heterotrimer association to the membrane is facilitated by binding of the Gα subunit to the Gβγ dimer, loss of γ7 prevented upstream receptor recognition and subsequent membrane association and was therefore sent for protein degradation. Thereby preventing G-protein-effector coupling following receptor activation. Further arguing against transcription as being the sole mechanism responsible for the selective 25 assembly of Gαolfβ2γ7, the analysis also revealed g2 as being more abundant than g7 in the striatum. Moreover, targeted knockout of G αl-/- in rat striatum does not lead to a reduction of β2 and γ 7 proteins (schwindinger et al.,2010). These results demonstrate the hierarchical order of heterotrimer assembly in rat striatum that is governed by the γ7 monomer in a post-transcriptional mechanism. Indeed, these studies identifying distinct Gαβγ combinations responsible for specific physiological functions may provide a basis to explain biased agonism and functional selectivity. Moreover, these studies provide a basis for our hypothesis that individual Gγ subunits can alter signaling to downstream effectors. 4.3. The g3 subunit Gγ3 is encoded for by the Gng3 gene found on chromosome 11 locus p11 and contains 2 exons in humans (Hurowitz 2000). Gγ3 is widely expressed throughout the brain in many organisms and share around 80% amino acid homology with Gγ2 and Gγ4. Zebrafish Gγ3 subunit shares a high degree of homology with humans and other mammalian Gγ3 subunits. Of all the Gγ proteins, the zebrafish Gγ3 protein is most closely related to its mammalian homolog sharing a 93% identical polypeptide sequence (Kelly 2001, 2008). The regional distribution of Gγ3 in the mammalian brain was first identified in 1997 and later in 2008 using immunohistochemical analysis of human (Morishita 1997) and rat brain tissue. While Gγ3 is ubiquitously expressed in the developing CNS and neural crest, these data provide a basis for our hypothesis that gng3/- fish may have an altered startle in response to acoustic stimuli. These studies revealed strong localization of Gγ3 in the neuropil and inner ear, with little expression in the neuronal cell bodies. Importantly, it was also observed that the levels of G γ 3 increased 26 in the developing CNS and neural crest and that these levels decreased in humans with old age. In 2001, Kelly et al., used whole-mount in situ hybridization and RT-PCR to determine the expression profile of Gγ3 during zebrafish embryogenesis. Gγ3 was first detected at 18-19hpf (late somitogenesis) where it was preferentially expressed on the Cranial nerve V. Preferential expression of G γ 3 was detected in distinct neuronal populations of the fore-, mid- and hindbrain. These signals were localized to the developing neural tissue and appeared to follow a similar expression profile to GABA and Acetocholinesterase (AChE) expressing neurons. Importantly, overexpression of B2 γ 3 resulted in defects in eye and forebrain development. Thus, the spatial and temporal expression pattern of G γ 3 indicates a possible role in transducing signals in the developing nervous tissue (Kelly et al.,2001) The first example of a gene silencing approach used to determine function of an individual g subunit occurred in 1993 when Kleuss et al., used gng3 specific anti-sense oligonucleotides in a rat pituitary cell line to show a requirement for G γ 3 in mediating Ca2+ influx through L-type Ca2+ channels in somatostatin and muscarinic receptor signaling (Kleuss C et al.,1993). Using a similar gene targeting approach, Macrezlepretre 1997, demonstrated that angiotensin induced activation of At1 receptors and the subsequent increase in intracellular calcium was dependent on G α 13/ β 1/ γ 3-effector coupling. Moreover, this study demonstrated that knockdown of any one of the G α 13/β1/γ3 G protein monomer abolished Gαt1 mediated increases in intracellular calcium (Macrez-lepretre 1997). 27 More recently, the Gng3(-/-) phenotype in murine models has been implicated in effecting opioid signaling, likely by altering the mu-opioid (Oprm1) signaling cascade (Schwindinger et al., 2004 , Schwindinger et al., 2009). In the first set of experiments conducted by Schwindinger, et al., investigators used a gene deletion method that resulted in the generation of Gng3-/- mice. When compared to the control group, the Gng3 -/- group presented with an increased seizure susceptibility (when induced by an 85-95 dB sound for 10 seconds), and a higher mortality rate. Female Gng3 -/- mice showed a reduction in weight gain, decreased adiposity and lower leptin levels when compared to female controls. These results led the researchers to the conclusion that the Gng3-/- phenotype exhibits both neurological and metabolic abnormalities. In line with their previous findings, Schwindinger, et al., sought to determine whether the lean phenotype observed in Gng3-/- mice may be a result of defective Oprm1 signaling. A high fat diet was fed to both treatment and control phenotypes. Gng3 -/mice show resistance to high fat diet-induced weight gain compared to control. Gng3 -/mice also showed reductions in both acute and chronic morphine responsiveness in addition to increases in mRNA levels of encephalin (Penk) in reward-specific brain regions in the midbrain. It is important to note that no differences in Cb1 stimulated GTPγS binding or AC activity was observed in the Gng3-/- mutants. However, the sole purpose of this study was not to characterize Cb1 signal transduction, and researchers only looked at specific reward-related brain regions. 28 Methods 1. Plasmid and bacterial protocols The human Cb1 receptor variant 1 (CNR1) and the human wild-type Gγ3 (GNG3) were obtained as clones in pcDNA3.1+ at Kpn I (5’) and Xho I (3’), and at EcoR1 (5’) and Xho I (3’) (cDNA Resource Center, Bloomsburg, PA), respectively (Figure 3). The pcDNA3.0 vector encoding EGFP was purchased from Addgene (Watertown, MA). All cloned genes were expressed under the mammalian CMV promoter and plasmids were selected for using an ampicillin resistance marker (Figure 4). Luria-Bertani (LB) broth and agar (Sigma-Aldrich, St. Louis, MO) was prepared by dissolving 25g, and 20g of LB-broth and LB-Agar powder per 1L of water. Solution was sterilized and melted by autoclaving at 121 C and 15 PSI for at least 15 minutes. LB was allowed to cool and supplemented with 100ug/mL of ampicillin (Sigma-Aldrich) for broth and agar plates. Competent 5-alpha High-Efficiency E. coli cells (New England Biolabs, Ipswich, MA) were used to amplify plasmid DNA (pDNA) according to manufactures protocol. In brief, competent cells were allowed to thaw on ice for 10 minutes followed by the addition of ~500 ng of plasmid DNA (pDNA) to the tube. The cell solution was then heat shocked by incubating the tube at 42°C for precisely 30 seconds and stored on ice for 5 minutes. The solution was diluted with SOC media and allowed to incubate at 37°C for 60 minutes while shaking at 250rpm. Two 10-fold serial dilutions in SOC media were performed on the mixture before plating onto several LB-Ampicillin selection plates. The plates were then incubated at 37°C for 24 hours and checked for single colony formation. An inoculation loop was flame sterilized and used to isolate a single bacterial colony. The 29 colony was transferred to a conical tube (USA Scientific, Orlando, FL) containing LB+ampicillin (100ug/mL). The liquid cultures were incubated at 37°C for 24 hours in a shaker incubator set to 250 RPM and were used to generate concentrated plasmid stocks. Liquid cultures were used to generate glycerol stocks for archival and long-term storage of transformed bacteria. A 50% glycerol solution was prepared by combining equal parts molecular biology grade glycerol (Sigma-Aldrich) and sterilized deionized H2O. Equal parts of bacterial liquid culture and glycerol solution were mixed in a cryovial and stored at -80°C. According to the manufacturer's instructions, the GenElute plasmid miniprep kit (Sigma-Aldrich) was used to extract plasmids from liquid bacterial cultures. In brief, 5ml of the overnight recombinant E. coli culture was pelleted at 12,000 x g for 1 minute, and the supernatant was discarded. The pellet was resuspended in the supplied resuspension solution and vortexed until a homogenous solution was achieved. Lysis was achieved by the lysis solution to the mixture and carefully mixing it. Lysis was allowed for 5 minutes at room temperature using the supplied lysis solution. Lysis was neutralized by adding the neutralization solution. The cell debris were precipitated and pelleted via centrifugation at 12,000 x g for 10 minutes. Next, the lysate was transferred to an assembled nucleic acid binding column and centrifuged at 12,000 x g for 1 minute. The flow-through liquid was discarded, and the column was washed with the supplied EtOHdiluted wash solution and centrifuged at 12,000 x g for 1 minute. The flow-through was discarded, and the column was allowed to dry by centrifugation at 12,000 x g for 2 minutes. Next, the binding column was transferred to a collection tube. The DNA was eluted from the column using the elution solution centrifuged at 12,000 x g for 5 minutes. 30 The concentration and purity were quantified using a NanoDrop-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific). 2. Ethanol precipitation of pDNA products Plasmid DNA was ethanol precipitated from solution to obtain a final [pDNA] of 0.5 ug/uL and A260/A280 between 1.8-1.9. From each sample, a 1uL sample was used to measure DNA concentration and purity via Spectrophotometry with a NanoDrop-1000 UV-Vis Spectrophotometer. For each ethanol precipitation reaction, 0.1 volumes of 3M Sodium-Acetate (Sigma-Aldrich), and 2.5 volumes of cold 100% EtOH was added to the DNA solution to achieve a final salt concentration of 0.3M and EtOH of 70%. The solution was mixed briefly and centrifuged at 20,000 RPM for 10 minutes at 4°C. Next, the supernatant was removed and the pellet was washed with 70% EtOH and centrifuged at 20,000 RPM for 5 minutes at 4°C. The pellet was allowed to dry and resuspended in the appropriate volume of solution to obtain a final [pDNA] of 0.5 ug/uL, Following the resuspension of the precipitated DNA, a 1 µL sample was used to verify concentration and purity using a NanoDrop Spectrophotometer. 3. Restriction enzyme digestion Plasmid identity was verified by restriction enzyme digest. The plasmid map was examined for restriction sites with the webtool NEBcutter V2.0 (NEB). For each digestion, 500-700 ng of pDNA was digested with the appropriate restriction enzyme. pDNA containing gng3 was digested with XHO1 and HINDIII using the 10 NEB2.1 buffer. pDNA containing Cnr1 was digested with XhoI and EcoR1 using the 1x NEB2.1 buffer. pDNA containing EGFP was digested with XmnI using the 10x CutSmart buffer (NEB). The digestion mixtures were heated to 37°C for 1 hour, and the enzymes were 31 inactivated by heating to 65°C for 15 minutes. The resulting digest was run on a 0.8% agarose gel and imaged using a ChemiDoc imager (BioRad, Hercules, CA) at 320 nm wavelength and verified for the presence of the corresponding insert. 4. Genomic DNA Isolation from Danio rerio According to manufactures instructions, genomic DNA Isolation from Danio rerio was isolated using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma). All zebrafish embryos and juveniles were stored at 4°C before isolation. More than 25 mg of tissue was suspended in Lysis T solution supplemented with 20 mg/mL of proteinase-K, vortexed thoroughly to break up tissue, and digested at 55C for 3 hours. Samples were removed and vortexed every 30 minutes for ~15 seconds. Following incubation, samples were treated with RNase A at room temperature for 2 minutes. Lysis was achieved following the addition of Lysis C solution and an incubation period of 10 minutes at 70°C. 100% Ethanol was added to the lysate. The Lysate was then transferred to a preassembled binding column and centrifuged at 12,000 x g for 1 minute. The flowthrough was discarded, and the lysate was loaded into the column. The spin column was washed twice with the supplied wash solution. The spin column was transferred to a new collection tube, and Elution Solution was added to the column and centrifuged for 1 minute at 8,000 x g to elute the DNA. The purity and concentration of the eluant were quantified using a Nanodrop Spectrophotometer at wavelengths of 260/280 nm. 5. RNA and cDNA preparation RNA was extracted from CHO-K1 cells and Rat brain (positive control) using the TRIzol reagent (Sigma-Aldrich) to determine the expression of Cnr1 and Gng3. CHO-K1 cells were plated on a 60mm culture dish 3-4 nights before RNA extraction. When cells 32 reached 80-90% confluency, the cell medium was aspirated, and the TRIzol reagent was added directly onto the cell monolayer. After a 5-minute incubation period at room temperature, cells were pipetted up and down to lyse cells and placed into a 1.5 mL centrifuge tube. For RNA extractions using rat brain, 50-100 mg of tissue was suspended in 1 mL of TRIzol. The sample was homogenized on ice using a cordless motor pellet pestle (Sigma). Chloroform (Sigma) was added directly to the tube at a 1:5 chloroform:TRIzol ratio and centrifuged at 12,000 x g for 15 minutes at 4°C. The upper aqueous phase containing the RNA was removed from the sample and transferred into a new 1.5 mL tube. To precipitate the RNA from the aqueous phase, 100% isopropanol (NAME) was added into the tube at a 1:2 Isopropanol:TRIzol ratio and left to incubate at room temperature for 10 minutes. Next, samples were centrifuged at 12,000 x g for 10 minutes at 4°C. The supernatant was removed from the tube, and the RNA pellet was washed with 75% ethanol at a 1:1 TRIzol:Ethanol ratio. The sample was vortexed briefly and centrifuged at 7,500 x g for 5 minutes at 4°C. The supernatant was removed, and the RNA pellet was left to air dry for 1.5 hours. The RNA pellet was resuspended in nuclease-free water. The concentration and purity of the RNA samples were measured using a NanoDrop Spectrophotometer at wavelengths of 260/280. First-strand cDNA was prepared from 1ug of total RNA primed with Oligo (dT23 using 1x ProtoScript Reverse Transcriptase and 1x ProtocoScript II reaction mix (NEB). The reaction was initiated by incubating the samples at 42°C for 1 hour and inactivated at 80°C for 5 minutes. 6. Single guide RNA (sgRNA) design 33 The UCSC Genome Browser (http://www.genome.uscs.edu/) was used to locate the genomic DNA sequence of gng3 from the Zebrafish Assembly May 2017 (GRCz11/danRer11). The DNA sequence was used to design a single guide RNAs (sgRNAs) to target the second coding exon, exon 3, of the g3 subunit (Figure 5). The regions containing the sequence of interest were entered into the CRISPR Genome editing tool from Integrated DNA technologies (IDT; https://www.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM). The sgRNA sequences were chosen based on the ones that had the highest efficiency on target score and off target scores. The PrimerQuest Tool from IDT was also used to design genomic DNA primers that flanked the Cas9 cut site. 7. Polymerase Chain Reaction PCR amplifications were performed in 25uL reactions with approximately 40-120 ng of DNA, 1x Taq DNA polymerase (Takara bio ), 1x Taq Reaction Buffer (Takara bio), 0.2mM dNTPs (sigma), 20 uM of forward and reverse primer (IDT) in nuclease-free water. All reactions were performed with a negative control by replacing DNA with nuclease-free water. PCR conditions were optimized to produce a single band by adjusting the concentration of DNA, annealing temperature, and number of cycles. PCR conditions and primers are shown in Table 3. 8. Agarose gel electrophoresis 1.5% agarose gel electrophoresis was performed in 1x TAE buffer (SigmaAldrich). PCR products and water blanks were resuspended in 1x purple Gel Loading Dye (New England Biolabs) and loaded into the gel. The gel was run for approximately 34 30 minutes at 100 volts. Gels were imaged using a ChemiDoc imager (BioRad) at a wavelength of 320 nm. 9. Sequence analysis Verification of gene targeting was assayed using amplification by PCR followed by Sanger sequencing and deconvolution analysis. Intron-spanning primers AP07-AP08 were designed to flank the Cas9 cut-sites to allow for amplification of the target region (Primer table). Genomic DNA extracted from the crispants and amplified using primers AP07-AP08 was run on a 1.5% agarose gel. The gel was analyzed for heteroduplex banding or smearing at the area corresponding to the size of the amplicon. These samples were selected to be sequenced. According to the manufacturer's instructions, PCR products were treated with ExoSAP-IT (NEB) at a Ratio of 5:2 PCR product:ExoSap to remove the residual dNTPs and primers. Briefly, the samples were diluted in the ExoSAP-IT and incubated at 37°C for 15 minutes. The ExoSAP-IT was inactivated by incubation for 15 minutes at 80°C. Each sample was mixed with the forward or the reverse primer and sent to GENEWIZ (South Plainfield, NJ) for sequencing. The results were analyzed using the Interference of CRISPR Edits (ICE) tool (Synthego; https://ice.synthego.com/). 10. Fura-2AM Calcium Assay G-protein coupling to the receptor complex was characterized using fluorophores that bind to calcium presumably released from the ER, a key feature of a GPCR inducing a Gq-like response. Culture media was removed, and cells were loaded with 7.5uM Fura2AM in DMEM without phenol red. Cells were incubated in the loading solution for 1 hour at 37°C and 5% CO2. Cells were then washed twice with 1ml of basic salt solution 35 (BSS). BSS is comprised of 130mM of NaCl, 5.4mM of KCl, 5.5mM glucose, 2mM of CaCl22H2O, 20mM HEPES, and 1mM MgCl2 that was adjusted to a pH of 7.2 by titrating 10M NaOH. After the cells were loaded, the solution was removed from the cell monolayer, placed in 1mL BSS, and incubated at room temperature in the dark for 20 minutes. Cells were then placed on the calcium imaging system, and background fluorescence was collected for at least 30 seconds. The time was marked, and cells were stimulated with 1mL of 10uM AEA (Cayman). Calcium was measured using an InCyt Basic Fluorescence Imaging System, kindly provided by Dr. Robert Aronstam, and acquired from Intracellular Imaging of Cincinnati, Ohio. An Olympus Uis2 20x objective, acquired from Olympus Corporation of the Americas of Shinjuku, Japan, was used with the imaging system to measure the changing ratio of 340nm and 380nm wavelength emitted by the bound and unbound Fura-2AM. Fluorescent intensity was measured at 510nm. 11. Cell culture and Transfections Chinese Hamster Ovary (CHO) cells (ATCC) were cultured in Dulbecco's high glucose modified medium (Sigma) supplemented with 10% fetal bovine serum (FBS), 0.5mM L-glutamine (sigma), 0.1 mM sodium pyruvate (Sigma) and 1% penicillin/streptomycin (P/S) in non-pyrogenic 60-mm tissue culture-treated dishes (USA scientific). Cells were maintained at 37°C and 5% CO2 in an incubator and kept at a low passage rate. The cells were sub-cultured every 4 or 5 days when 80-90% confluency was reached. To subculture cells, the growth medium was aspirated, and the cells were rinsed twice with 1x Phosphate-buffered saline (PBS) (sigma). Cells were detached using 0.25% trypsin, 1 mM EDTA (Thermo Fisher Scientific) and seeded into a new dish. Cells were 36 cryopreserved and frozen at a slow rate of -1°C per minute using Nunc cryovials and culture media supplemented with 5% dimethylsulfoxide (DMSO) (Sigma) in a -80°C storage freezer. Cells were transferred and stored in vapor-phase liquid nitrogen for archival and long-term storage. For experiments involving the expression of Cb1, gng3, or EGFP, cells were transiently transfected using the electroporator according to manufactures instructions. In brief, cells were seeded into a 60mm dish with DMEM+10%FBS+1%P/S two days prior to transfection. Cells were washed twice with an equal volume of PBS and detached from plates using a 0.1% Trypsin-EDTA solution after reaching 75-85% confluency. For all transfections, 1x10^6 cells were pelleted and resuspended in nucleofection solution (Lonza). The cell suspension and 2.0 ug of pDNA were added into the cuvette and transfected using the H-014 nucleofector program. Immediately following electroporation, cells were transferred into 600ml of pre-equilibrated DMEM. pcDNAEGFP was used as a positive control to confirm that the nucleofection had been successful and as a standardized variable to keep the concentrations of DNA for transfection the same. 12. cAMP measurement G-protein coupling to Adenylyl cyclase was measured using the cAMP-Glo™ Luminescence Assay (Promega) following manufactures instructions. In brief, transiently transfected CHO-K1 cells were seeded into a 96-well black plate (Thermo) at a density of ~6.25x10^4 cells/well in 200ul of growth medium. Immediately prior to assaying, the medium was aspirated and cells were washed with twice with phosphate-buffered saline solution to remove traces of serum. Cells were then treated with 20uL of their respective 37 1x agonist (ANA, FSK, ANA+FSK, and Induction buffer) for 10 minutes at room temperature using a shaking plate (Thermo). Next, lysis of the cells was achieved by adding the supplied cAMP-Glo™ Lysis Buffer and incubating for 20 minutes at room temperature on a shaking plate. Then, the PKA reagent containing the PKA substrate and holoenzyme (cAMP-Glo™ Reaction Buffer) was dispensed into each well . The kinase reaction was carried out for 20 minutes at room temperature. Finally, an equal volume of Kinase-Glo Reagent (80uL/well) was added and allowed to incubate for 10 minutes. Luminescence was read using a BioRad plate reader. To establish a semi-quantitative measurement of cAMP, the DeltaRLUvalues were calculated by subtracting the average RLU for each triplicate by the average RLU value of cells treated with the induction buffer. Agonist preparation: Agonists were freshly prepared the morning of each experiment. 2ArachadonylEthanolamide (Anandamide) was purchased from Cayman Chemicals (Batch #0525560-30) at a stock concentration of 14 mM. A 4x working solution was prepared by removing 1uL of the stock solution and diluting it in 3.6 mL of PBS. The solution was then diluted in the induction buffer containing PBS and 1 mM isobutyl-1methylxanthine (IBMX) (Enzo), a potent phosphodiesterase inhibitor, to obtain a 1 uM concentration used for treatment. Forskolin was obtained from EMD Millipore Corp, USA, (lot#3387887) at a stock concentration of 50mM. A 4x working solution was prepared by removing 1uL of Forskolin and diluting it into 250uL PBS. The solution was then diluted in the induction 38 buffer containing PBS and 1 mM IBMX to obtain a 50uM concentration used for treatment. 13. Fish care and Embryo rearing All protocols involving animals were approved by the Bloomsburg University of Pennsylvania Institutional Animal Care and Use Committee (IACUC #172). Adult fish (The Fish Place, Lancaster PA) were maintained in our facility's freshwater fish room. After purchasing, fish were quarantined for two weeks, and the tank water was treated with a prophylactic dose of Microbe-Lift (Ecological laboratories) and NITE-out (Ecological laboratories). Initially, the water chemistry was checked daily using an API Freshwater Master Test Kit. After water quality remained stable for two weeks, water chemistry was monitored daily. Water pH was maintained between 7-8, alkalinity between 50-100 mg/L, hardness at least 75 mg/L, and nitrogenous waste less than 0.02 mg/L (Harper and Lawrence, 2011). Fish were maintained on a 14:10 day/night light cycle to induce spawning. Water temperature was maintained at 28°C, and an air pump was used to oxygenate the water. Tank water was filtered through a reverse osmosis/deionized (RO/DI) filtration system (Spectrapure) and delivered automatically to each aquarium from a holding tank. A water conditioner (Aqueon) was added as needed. 14. Spawning and Embryo Rearing: Fish used for breeding were between 7-12 months of age and were housed together in the same tank. Crosses were set up the evening prior to embryo rearing by placing single male and female fish separate in a divided breeding tank. The dividers were lifted 20 minutes prior to the lights turning on. The fish were given approximately 39 20 minutes to spawn before embryos were collected. If no embryos were present at 20 minutes, fish were allowed an additional 20 minutes to spawn. 15. Validation of a behavioral paradigm to measure startle and locomotor activity after sound challenge. Zebrafish juveniles (7dpf) were placed individually in wells (2inx2in) containing 6ml of 1x egg water or 1x egg water treated with either low dose ANA (10uM) or highdose ANA (40uM). Doses were modified from those used by Sufian et al., 2018. Juveniles were submerged in their appropriate solution for a treatment period 15 minutes prior to assaying and remained in the solution throughout the experiment. A video camera (12MP iPhone 13 Pro Max) was mounted above the assaying dish on a ring stand. Fish were recorded for 2.5 minutes to establish a baseline activity level before a brief acoustic stimulus of 120 Db (Shoreline Marine Airhorn) was delivered 1 meter away from the experimental setup. Fish were then recorded for an additional 2.5 minutes to measure locomotion. The video was imported into VLC and frames were extracted (10 frames/second). (Note: This step was to reduce the frame rate to a manageable amount that was used for a frame-by-frame analysis.) Frames were then imported into ImageJ and the video was converted into 8-bit grayscale and made binary by adjusting the threshold to create a clear distinction between the juveniles and the background. The region of interest was selected, and movement data was processed individually for each well. The area of interest and x and y coordinates were imported into excel for analysis. 16. Validation of a Behavioral assay to measure startle and locomotor activity after light challenge 40 Zebrafish juveniles (7dpf) noninjected wild-type or juveniles injected with GFP were placed individually in wells (2inx2in) containing ~4mL of 1x egg water. Fish were allowed to acclimate to their new environment for 10 minutes with the lights on high intensity. A video camera (12mp iPhone 13 Pro Max) was sitting 18 inches above the assaying arena. The fish were recorded for a total of 16 minutes: 4 minutes with the lights on high intensity to establish baseline locomotion, 4 minutes with the light at low intensity (dark challenge), and then 8 more minutes with the lights on high intensity. The video was imported into VLC and frames were extracted (2 frames/second). The frames were divided into the 3 phases (Light:Dark:Light , 4min:4min:8min) and imported separately into imageJ. The video was converted into 8-bit grayscale and made binary by adjusting the threshold to create a clear distinction between the juveniles and the background. The region of interest was selected, and movement data was processed individually for each well. The Area of interest and X and Y coordinates were imported into excel for analysis. 17. Preparation of CRISPR-reagents for Microinjection The Alt-R CRISPR-Cas9 system was purchased from IDT. The Alt-R crRNA (guide) and tracrRNA was supplied as a lyophilized powder and were resuspended in the appropriate volume of Nuclease-Free IDTE buffer to reach a final working concentration of 100uM. Assembly of the Ribonucleoprotein complex occurred in the mornings within 1 hour before injections. First, a 3uM gRNA solution was assembled by combining 3uL of 100uM Alt-R CRISPR-Cas9 crRNA, 3uL of 100uM Alt-R CRISPR-Cas9 tracrRNA, 94 uL of Nuclease-Free Duplex Buffer, and heated for 95 C for 5 minutes. Next, the Cas9 protein (10ug/uL) was diluted to a working concentration of 0.5 ug/uL by combining 41 0.5uL of the Cas9 protein with 9.5uL of the Cas9 working buffer (20mM HEPES; 150mM KCL, pH7.5) Note: The increase of ionic strength and addition of KCl has been shown to increase the solubility of the Cas9 protein and increase cutting efficiency (Burger et al., 2016). Finally, assembly of the RNP complex was accomplished by combining 3uL of the gRNA with 3 uL of the diluted Cas9 protein and incubated at 37°C for 10 minutes. 18. Microinjection of CRISPR-components into Zebrafish Embryos All microinjections were conducted using a Narishigi Nikon Micromanipulator/Microinjection system (Model IM-9b). Calibration of the machine was required to determine the amount of injection solution was being delivered. We used a calibration micrometer slide (Amscope) with a large drop of mineral oil placed over the scale. Methylene blue was backloaded into the needle and injected into the oil droplet. This was repeated until a consistent 0.15 mm droplet diameter (contains approximately 2nL-3nL of solution) was achieved. Embryos were collected using a 1ml plastic pipette, washed with 1x egg water containing 60 ug/mL of instant ocean salt in sterilized dH2O, and transferred to a 10cm petri dish (Thermo) filled with room temperature egg water. Approximately 10-15 embryos were lined up against a glass slide in a petri dish and viewed under low magnification to ensure that the embryos were not developed past the 2-cell stage (Figure 6). It is recommended that the microinjection solutions contain the Cas9 protein and sgRNA in a 2:1 ratio of Cas9:sgRNA to obtain a final concentration of 400pg/nL of cas9 protein and 200 pg/nL of sgRNA and injecting 1nl of solution (sorien, et al.,2018). For our experiments, we used a final concentration of 103 pg/nl sgRNA (crRNA is 36 ng/uL and 67 ng/uL 42 tracrRNA) and 0.5 ug/ul of Cas9 protein and delivered ~2nl of solution into the embryo yolk sack. The solution contained 0.08% Phenol red for visual confirmation of the injection. Following the injection, embryos were returned to their incubator tank to develop. Embryos were imaged daily using to inspect the health, calculate survival and remove the dead embryos from the tank. A 1mm capillary tube (World Precision Instruments) was pulled using a P97 needle puller (Sutter instruments) We used the following program to create a tip suitable for injection: Pressure = 400, Heat=512+Ram, Pull= 125, Vel= 075 , Time=200. The tip of the needle was swiped with a KimWipe (Fisher) to obtain an angled opening that could easily pierce the chorion. 43 Results 1. Expression analysis of gng3 and Cnr1 in CHO-K1 cells To establish a model system that will allow me to analyze the effect of the forced expression of GNG3 and CNR1, the endogenous expression levels of Gng3 and Cnr1 were determined by performing PCR on the cDNA synthesized from RNA extracted from CHO-K1 cells, and Mesocricetus auratus (hamster) brain as a positive control. Gng3 is most abundantly expressed in the brain, while Gng10 is more ubiquitously expressed throughout many tissue types (Syrovatkina et al., 2016). Moreover, Cnr1 is also widely distributed in many tissue types. Following the optimization of PCR conditions, primer pair AP03-04, WS93-94 and WS99-100 were used to amplify the prepared cDNA from hamster brain and CHO-K1 cells to determine the expression profiles of Cnr1, Gng3 and Gng10, respectively. Our results show that the gng3 and Cnr1 transcripts are expressed in hamster brain but not in CHO-K1 cells . The Gng10 transcript was present in both hamster brain and CHO K1 cells (Figure 7). This makes CHO-K1 cells an ideal model system to use for identifying the role that gng3 plays in Cnr1 mediated signal transduction. 2. Concentration of DNA for transfection The concentration of the pDNA is important for optimal transfection efficiency. For transfections using the Amexa Electroporation system, an OD 260/280 of 1.7-1.9 and a concentration of DNA that would allow for 2ug plasmids/sample in 4uL of water is recommended by the manufactures. To accomplish this, the initial pDNA concentration and purity was determined using a NanoDrop1000 (data not shown). The pDNA was 44 cleaned and concentrated using a sodium acetate-ethanol precipitation to reach a final pDNA concentration of ~500ng/ul and OD of 1.7-1.9 . Transfection efficiency via electroporation was determined by co-transfection of a plasmid that expresses EGFP in parallel to cells transfected with Cb1 and Gng3 in all assays. 3. Restriction Enzyme Digest A restriction enzyme digest was used to verify the identity of the plasmids that were isolated from E. coli. Gng3 and Cnr1 were cloned into the pcDNA3.1+ vector and EGFP was cloned into the pcDNA3 vector (Figure 4). Inserts corresponding to the expected product lengths were visualized on a 1% agarose gel stained with EtBr (Figure 8). 4. Validation of the cAMP-Glo assay Cannabinoid receptor agonist-mediated inhibition of Fsk stimulated cAMP accumulation in a classical Gi/o dependent manner is a hallmark trait of the Cb1 receptor. While there are different ways to measure cAMP accumulation, we chose a PKA-dependent reporter assay that is dependent on the concentrations of [ATP]. A cAMP standard curve was generated at concentrations ranging from 0-4uM. Standard curves were also generated in other 96-well plates to determine which was best suited for assaying. Standard curves were generated using 96-well black plates were linear at [cAMP] ranging from 0-0.125 uM (Figure 11). 5. Does forced expression of gng3 alter cAMP accumulation in CHO-K1 cells? CHO-K1 cells treated with Fsk, AEA, FSK+AEA, and a vehicle. An Analysis of Variance reveled no significant difference between treatment groups (Pvalue=0.6286), 45 Thus, we reject our hypothesis that forced expression of Gng3 and Cb1 in CHO-K1 cells will significantly alter cAMP accumulation when compared between treatment groups. While no statistically significant differences exist between treatments, it is important to note an obvious trend between cells transfected with Cb1 or Cb1+GFP and treated with Anandamide. CHO-K1 cells expressing only the Cb1 receptor and treated with AEA trended towards decreasing cAMP accumulation (when compared to basal cAMP). However, when co-transfected with gng3 and treated with AEA—cAMP accumulation tended to increase above basal levels. 6. Measurement of intracellular calcium transients The ability of Cb1r agonists to inhibit synaptic transmission through the modulation of intracellular calcium transients is mediated by a variety of canonical and non-canonical G-protein mechanisms. Studies using primary cell lines derived from different types of neural tissue like Astrocytes (Hegyi,2018,), Rat Cerebellum (Daniel, et al., 2004) and neuroblastoma cells (Sugiura 1999) demonstrate the ability of Cb1 to activate IP3 release and subsequent release of intracellular Ca2++. In contrast, other studies using CHO cells expressing Cb1 have failed to detect this response. Since the g3 subunit is preferentially expressed in the brain and has been found to be colocalized in Cb1 expressing cells, we hypothesized that CHO-K1 cells expressing the g3 subunit could evoke the release of intracellular calcium when stimulated with Anandamide. We tested this by stimulating CHO-K1 cells transiently expressing recombinant Cb1 and Cb1+gng3. Both groups were treated with 10um and 100uM uM AEA and failed to show any change in intracellular calcium transits Leading us to reject our hypothesis that 46 forced expression of gng3 and activation of Cb1 will evoke the release of intracellular calcium stores when stimulated with Anandamide (Figure 10). 7. Validation of a VMR Assay in zebrafish A VMR assay was employed to measure the response of juvenile zebrafish in response to a light and dark challenges. After 10 minutes of acclimating to their new environment, WT juvenile zebrafish (7dpf, n=9) travelled 144±36 mm during 4 minutes under continuous high intensity lighting (Phase 1) and 108±32 mm during 4 minutes under continuous high intensity lighting (phase 3) following a period of 4 minutes at low intensity lighting (phase 2). While fish trended to travel less following low lighting conditions, these differences were not statistically significant (t test=0.217). The total distance traveled under low intensity lighting (phase 2) was not obtained for the WT group (Figure 11). Considering this paradigm was intended for use on our zebrafish mutants, we wanted to assure changes in VMR was not affected by our injections. A GFP plasmid was injected into embryos immediately after fertilization in parallel to our non-injected WT fish. GFP injected juvenile zebrafish (7dpf, n=8) traveled an average of 133±36mm in phase 1, 173±52mm in phase 2 and 104±37 mm during phase 3. Distance travelled by GFP mutants also tended to be less but was insignificant between phase 1 and 3 (ttest=0.200). However, distance travelled in phase 3 was significantly less than distance traveled in phase 2 (t-test=0.030). Moreover, our results show no significant differences between the distances WT and GFP in phase 1 (t-test=0.803) or phase 3 (T-test=0.911). Thus, our GFP injections did not have a significant impact on distance travelled. 47 8. Validation of an Acoustic Startle Paradigm in Zebrafish Larvae (7dpf) An acoustic startle assay was performed on zebrafish larvae, some of which had been treated with Anandamide (10uM). An air horn was used as the acoustic stimulus (120dB) and the distance traveled 0.33 seconds before the stimulus and 26.40 seconds after the stimulus was calculated. In trial 1, fish in the treatment group travelled and average of 3.246 mm (standard error=0.066mm) before the blast and 54.841mm after the blast (standard error=1.146mm). The control group in trial 1 travelled an average of 3.148 mm (standard error=0.098 mm) before and 57.747 mm (Standard error=1.385 mm) after the blast. Distance travelled in trial 1 was not significantly different between treatment and control before (T-test=0.904) or after (t-test=0.726) the blast. Total distance travelled in trial 1 was also not significantly different between the treatment and control groups (ttest=0.741). The average startle latency in trial 1 was 0.136 seconds for the control and 0.099 seconds for the treatment group and this difference was not statistically significant (T-test=0.26; Figure 12). In Trial 2, Fish in the treatment group travelled and average of 2.602mm before (standard error=0.0843) and 35.591mm (standard error=0.0843secs) after the blast. Whereas, the control group traveled an average of 3.554 seconds before (standard error=0.146) and 43.147 seconds (standard error=1.243 seconds) after the blast. The distance travelled in trial 2 was not statistically significant between treatment and control groups before (t-test=0.421) or after (t-test=0.376) the blast. The total distance travelled in the experiment by the treatment vs control groups was also not significantly different (t test=0.357; Figure 12). 48 To evaluate the reliability of our assay, tests for significance between trial 1 and trial 2 were conducted. Distance travelled between treatment and control in trial 1 and 2 before (t-test=0.536)and after (t-tests=0.356) the blast, and in total (t-tests=0.344) were all insignificant (Table 4). This data suggests that are data was reliable between 2 trials and allows us to reject our hypothesis that the distance travelled in zebrafish larvae would be significantly affected by treatment with anandamide (10uM). Significance in startle latency was analyzed by a Chi2 test between the frame the fish first startled (moved two std. deviations above the average distance) showed no difference between treatment and control groups ( X2 = 24.778, df = 25, p-value = 0.4749). Histograms of the distribution is shown in Figure 13. 9. Microinjections and CRISPR/Cas9 Primers were designed to amplify a 350bp amplicon that flanked the cut site (AP11-12). Genomic DNA was isolated from the crispants and amplified using primer pair AP11-12. The results in Figure 15 reveal a banding pattern that is consistent with an indel formation. These samples were then sent out for Sanger sequencing and chromograms were analyzed with ICE (Synthego) to determine genomic editing efficiency. Sample 2-1 had the lowest editing efficiency with 12% (R2=0.98) of the total alleles containing a 4bp deletion and less than 1% with a 10bp deletion. Sample 2-5 harbored the highest formation of indels with an editing efficiency of 93% (R2=0.99) of the total alleles containing a 4bp deletion. Sample 2-6 had an editing efficiency of 72% (R2=0.98) of the total alleles containing a mix of both insertions and deletions. 49 Discussion 1. Expression analysis of Gng3 and Cnr1 in CHO-K1 cells One of the primary objectives in this study was to understand how Gg3 coupling to the Cb1 receptor effected the activation of downstream signaling cascades. To do this, we used a Chinese hamster cell line (CHO-K1) as a representative model to evaluate these effects in vitro. The expression levels were characterized by using RT-PCR on the mRNA isolated from lysed CHO-K1 cells. The cDNA obtained was amplified using primers specific to Cnr1, Gng3 and Gng10. Gng10 was amplified as a positive control for the PCR reaction and all PCR reactions were performed in parallel with cDNA obtained from hamster brain as a positive control for the primers. Our data indicate that CHO-K1 cells do not endogenously express the Gng3 or Cnr1 transcript but do express the Gng10 transcript. CHO cell lines are an epithelial cell line derived from the ovary in Chinese hamsters. They are a robust cell line that is often used in biological and pharmaceutical research because of their ability to produce recombinant proteins (Wurm et al., 2004). Moreover, CHO cells allow for post-translation modifications to recombinant proteins that are more similar to those in human cells, especially when compared to other on-the-market cell lines (Ghaderi et al., 2012). This was an important factor in our studies considering the post translational isoprenylation occurring to the Gg3 subunit. In contrast to other cell lines, CHO cells are amenable to several gene amplification techniques and have a well characterized genome (Tingfeng et al., 2013). Despite being a common method for measuring gene expression, our experimental design was limited by the basic premise of RT-PCR. Considering RT-PCR is based on 50 the ability of sequence-specific primers binding to mRNA transcripts before its translation into a protein, it is possible that Gng3 or Cnr1 was rapidly translated into a protein and thus not detected in our reaction. However, this would be unlikely considering that Gng10 was detected in the reaction and undergoes similar posttranslational modifications as Gng3. Considering that Cnr1 is intronless and therefore has one less major RNA processing event to skip over (Onaivi et al.,, 1999), this may have been more likely in Cnr1. While it is theoretically possible that the Gng3 and Cnr1 transcript underwent rapid translation and was therefore not detected using RT-PCR, it is extremely unlikely considering the high sensitivity of RT-PCR (Wong and Medrano 2018). This could have been reconciled by assaying for protein levels in parallel to our RT-PCR analysis. Additionally, it is possible that using only a single cell line may have interfered with us gathering more biologically relevant data. Future researchers may benefit from using both continuous and primary cell lines derived from populations of cells from humans or other organisms that they are interested in studying. 2. Concentration and Purity of DNA for Transfection Establishing an expression system suitable for assaying was an imperative step in our measurement of cAMP and intracellular calcium (discussed below). We established transient expression models of Cb1 and Gng3 in CHO-K1 cells via the electroporation of plasmids encoding our genes of interest. Plasmids were obtained through the cDNA Resource Center. We ensured all plasmids were at a A260/A280 between 1.7-1.9 prior to transfection. Transfection efficiency of at least 80% was validated in each experiment by co-transfection with a GFP expressing plasmid. GPF was visualized using fluorescent 51 microscopy. To check the identity of these plasmids, we used restriction enzymes that cut areas spanning our gene of interest. The restriction enzyme digest was electrophoresed on an agarose gel and the insert was compared to a ladder of known sizes (Figure 14). Both Cnr1 and Gng3 were cloned separately into pcDNA3.1. The plasmids encoding Cnr1 were cut with Xhol and EcoR1 to yield expected product lengths of 5427bp and 1419bp. We found bands present at lengths of 5450bp and 1400bp. The plasmids containing Gng3 were cut with Xhol and HindIII to yield expected products of 5408bp and 250bp. We found bands present at lengths of 5400bp and 250bp. The EGFP cloned into pdDNA3 was cut with Xmn1 and made cuts at position 2220 and 5601. The expected product lengths were 3381bp and 2778bp and bands were found at 2800bp and 3350bp. These data suggest that the plasmids used in our transfections contained our gene of interest. However, we did not conduct immunoblots to confirm expression. Transient transfections have often been used in studies aimed at evaluating gene functionality, especially in CHO cells (Muller et al., 2007). While there are a wealth of tools that allow researchers to heterologously express recombinant proteins, we chose to use electroporation because of its well validated track record for high-efficiency transfections (Kim, T. K., and Eberwine, J. H. 2010) . By co-transfecting GFP, we were able to visually confirm the delivery of the plasmid, a common method in cell transfection studies (Chicaybam et al.,2017). While transient transfections certainly have their use in pharmacological perturbation studies, they do not come without limitations. Transient transfections typically result in the expression of proteins at levels well above those in normal, physiological conditions (Blasi et al., 2021). We initially observed poor confluency of our CHO cells following 52 electroporation of our plasmids. Moreover, the CHO cells appeared unhealthy when visually inspected. Despite having an A260/A280 between 1.7-1.9 which indicated a relatively pure sample of plasmid DNA (pDNA), we attributed the poor growth to possible contamination from the E.coli used to amplify our plasmids. This was reconciled using an ethanol precipitation of our pDNA prior to electroporation. Precipitation of pDNA has been shown to increase transfection efficiency and reduce contamination Irwin and Gutmann (1997) . It should also be noted that that the precipitation also allowed us to achieve a pDNA concentration of ~500 ng/µl (as recommended by manufactured), which improved transfection efficiency and allowed better growth of the cells. It is important that future researchers using E.coli as a host for cloning take extra precautions to eliminate contamination prior to transfection. We found that using an ethanol precipitation of our pDNA increased transfection efficiency and optimized for better cell growth after electroporation. Moreover, researchers should consider establishing a stable expression cell line that is more representative in terms of gene expression to those in physiological condition. This should include measuring protein levels and comparing it to in-vitro levels of expression. 3. Validation of the cAMP-GLO Assay An important component for in vivo functional characterization of cannabinoid receptors is the ability to measure cAMP levels. We chose to utilize an indirect reporterbased method of cAMP quantification using the cAMP-GLO assay. Intracellular cAMP activates PKA which modulates the amount of ATP in the assay. The ATP levels are 53 coupled to a luciferase reaction and are quantified by the change in Relative Light Units (RLU’s) emitted from the sample (DeltaRLUs). Using known concentrations of cAMP in black, 96-well plates, we were able to generate a linear correlation at cAMP concentrations from 0-0.125uM. There are several methods to functionally characterize GPCRs in terms of cAMP quantification. While the standard method of quantitating adenylyl cyclase activity it to measure the conversion of [a-32P]ATP to [32P]cAMP, this method requires specialized equipment and approvals to work with radiolabeled atoms (Salomon et al., 1974, Zheng and Xien 2012). Nevertheless, PKA-dependent reporter assays have been shown to be a reliable method in studies using CHO cells (Kumar et al.,2007, Goueli and Hsaio) and are safer than radiolabeling studies. Utilization of the black 96-well plates was prompted due to the current availability of supplies at the time our research had occurred. While the manufactures recommended using white 96-well plates, supplies were extremely limited due to the COVID-19 pandemic (Woolsten, 2021). We had also measured standard cAMP concentrations in both V-bottom and clear plates with little success (data not shown). Luminescence from neighboring wells had interfered with our readings and prevented accurate measurement. Validation of this assay proved to be a major hurdle in our experiments. While black plates yielded the most accurate data of our standard, it created inherent complication in the visual confirmation of the number of cells present in each well. We reconciled this by plating the cells in clear plates in parallel to the black plates. The number of cells in clear plates were counted and verified before assaying. While this helped control for the number of cells in each well, it did not correct for the limitations that are associated with 54 reporter assays (Kain and Ganguly 2001) including their high sensitivity to any cell stress. Several experiments have shown that reporter assays are extremely amenable to many types of interference (Neefjes et al.,2021) and that the data generated can be highly variable amongst similar studies (Niedermnerg et al.,2003). While Delta RLU’s in our standard curves that were used to validate our equipment remained relatively constant, the majority of our issues presented when using lysed CHO cells. Despite our assaying system being sensitive to DeltaRLUs corresponding to cAMP concentrations in the low micromolar range (0-0.125uM), high luminescent signals present at random in the wells of untreated lysed CHO cells complicated our analyses. We initially attributed this to defective Protein Kinase A (PKA) from the manufacturer. This was reconciled by using new PKA from separate lot numbers (kindly provided by Promega Corp). While this did provide us with more consistent results, we still had encountered problems with high variability in luminescent signals when measuring cAMP in lysed CHO cells not receiving a treatment. Ultimately, we attributed the ranging levels of DeltaRLUs to the presence of high amounts of ATP in our cells. Considering that the ATP concentration in cells are influenced by many factors and that CHO cells are glycolytically active and thus have a high rate of ATP turnover (Zhang et al., 2021), any small changed in the initial culture conditions can dramatically alter the levels of intracellular ATP. This in turn can amplify the signals detected in each trial due to the mechanism of the luciferase assay. In retrospect, it would have been beneficial to serum starve the cells prior to assaying. This would significantly eliminate the amount of ATP that would interfere with the luciferase reaction. 55 If possible, future research should focus on quantifying cAMP by using methods that yield results that are more reproducible, like radioactive tracer assays. In instances where this equipment is not readily available, researchers should consider the mechanism used to detect cAMP in their assay to be sure that other protocols in their experiment (like methods of cell culturing or treatment times) will not interfere with the data that they obtained. 4. Forskolin-stimulated cAMP accumulation in CHO-K1 cells expressing gng3 and Cb1 The data presented in this study suggest that although cAMP accumulation is not significantly changed upon Gg3 forced expression, an obvious trend exists between cells transfected with a plasmid for Cnr1 or with plasmids for Cnr1 and Gng3 and treated with anandamide. CHO-K1 cells expressing only the Cb1 receptor and treated with AEA trended toward decreasing cAMP accumulation when compared to basal levels of cAMP. However, when expressing Cb1 and Gg3 and treated with AEA, cAMP accumulation increases above basal levels. These data suggest that the presence of Gg3 in Cb1 expressing CHO cells can change the cell from inhibiting AC-mediated cAMP accumulation to activating AC. Nevertheless, the lack of statistical significance and the high variation between trials lead us to reject our hypothesis that preferential coupling of the Gg3 subunit to the Cb1 receptor is responsible for increasing cAMP accumulation. While our data lacks statistical significance in the differences in cAMP accumulation when CHO cells are co-transfected with Gng3 and treated with AEA, it highlights an observation that has been reported on by several other independent researchers— pleiotropy of the Cb1 receptor. While activation of the Cb1 receptor predominately 56 couples to Gi proteins, several observations highlight the heterogeneity of this system and have led to speculations that the Cb1 receptor is promiscuous. Our experimental design logic was to determine whether the ‘forced’ coupling of Gg3 to Cb1 would have an effect on cAMP accumulation. We proposed that co-transfecting Cnr1 and Gng3 in cells that do not endogenously express these proteins would allow the Gg3 subunit to be the primary gsubunit in the G protein heterotrimer. Moreover, in cells only expressing Cb1, the heterotrimer would consist of Ga,Gb and Gg subunits that are endogenously expressed in CHO-K1 cells. Stimulation of Cb1 with its endogenous agonist AEA and measurement of cAMP would unveil a response that was mediated by either the inhibition or activation of AC. We thought that if we forced Gg3 to be the primary g subunit that associated with the receptor complex, then we would observe a ‘switch’ in responses. In other words, cells treated with AEA and expressing only Cb1 would decrease cAMP whereas cells expressing Cb1 and gng3 would increase cAMP. However, our experimental results proved to be more complicated as we experienced large ranges of DeltaRLU’s and often only subtle differences in the levels of cAMP. Despite this being the first study (to the best of our knowledge) looking at the role of the g subunit on activation or inhibition of AC (as measured by cAMP accumulation) for the Cb1 receptor, it is not the first study to identify Cb1s ability to ‘switch’ from decreasing to increasing cAMP. One study identified this switch in a subset of neurons in the globus pallidus of rodents (caballero et al.,2016). What started as an observation that cannabinoid treatment would sometimes enhance synaptic transmission in these dopaminergic neurons, Caballero et al. (2016) showed that blockade of Gi proteins by PTX treatment resulted in the increase of cAMP (mediated by Gs). Earlier evidence for a 57 Gs linkage to the Cb1 receptor occurred in 1997 when Glass and Felder demonstrated that concurrent stimulation of D2 receptors and Cb1 receptors in striatal neurons inhibited cAMP accumulation but when treated with PTX, cAMP accumulation increased. Furthermore, they showed that this response was mediated solely by the Cb1 receptor and occurred in a concentration-dependent manner that was blocked by Cb1 antagonist. Interestingly, these results were reproducible using CHO cells only expressing the Cb1 receptor (Glass and felder, 1997). The researchers in these aforementioned studies speculated that G proteins were being sequestered by a common pool of readily available G proteins, and that blockade of Gi via PTX unmasked the stimulatory properties of Cb1. In our study, it is possible that a large common pool of readily available G proteins are present in the cytoplasm. Considering we only experimented using a single GPCR and did not use toxins to disrupt the coupling of Gi proteins, g3 may not have been able to out compete other g subunits from being associated to the heterotrimer. In other words, Cb1 receptors can still interact with heterotrimeric G protein complexes that are formed without the g3 subunit. Moreover, by using only one GPCR, the Cb1 receptors could still interact with other heterotrimeric G protein combinations that contain an inhibitory α subunit and another g subunit. This in turn means that the inhibitory actions of the heterotrimeric complex would be competing with the stimulatory actions of the heterotrimeric G protein complex with g3 that we proposed, and its contribution was only minimal to the overall levels of cAMP. Perhaps a more appropriate experimental design would have incorporated simultaneous transfections with other GPCRs that could deplete the common pool of inhibitory G proteins, therefore allowing the stimulatory actions of Cb1 coupling to g3 to prevail. Similarly, we could have also treated the cells with PTX to 58 eliminate the inhibitory properties of the Gαi subunits. While these experiments may reconcile the possibility of Cb1 coupling to other net- inhibitory heterotrimeric combinations , we speculated that g3 would be more abundant considering that transient transfections typically result in proteins being expressed at levels above those under endogenous conditions (tubio et al., 2010). In support to the claim that the inhibitory actions of Gai were significantly greater than the stimulatory properties of Gg3, research conducted by (Saroz et al.,2019) demonstrated that stimulation of AC by Gbg in CB2 expressing cells occurred first, causing an initial increase in cAMP. This was then followed by a decrease in cAMP mediated by the Gbg dimer. This may suggest that inhibition and stimulation of AC may be occurring simultaneously through the actions of different AC isoforms (Saroz et al.,2019) which may result in an initial inhibition of AC through the actions of GaI. In the context of our experimental design, cAMP was only measured after the cells were stimulated with either FSK or AEA after 10 minutes. This would mean that our studies were limited to cAMP levels present at a specific time point thus, preventing us from observing the different phases in increasing levels of cAMP followed by decreasing levels of cAMP. In hindsight, a more appropriate experimental design would have been to incorporate treatments with agonists at several timepoints before measuring cAMP accumulation. The lack of significance in cAMP accumulation along with the large range of data obtained from our experiments of the same treatment group but in different trials ultimately lead me to conclude that the presence of Gg3 does not alter cAMP accumulation. However, it may be beneficial for future studies to first identify the 59 “primary” g subunit used in GPCR signaling and subsequently knocking out that subunit before experimenting. This will prevent the possibility of the canonical GPCR heterotrimer from being associated to the Cb1 GPCR complex. Moreover, an initial investigation onto what AC isoforms is present, and their selective responses to various g proteins could prove useful in any future experiments looking at G protein signal transduction. 5. Intracellular Calcium in AEA stimulated CHO-K1 cells expressing gng3 and Cb1 Our experiments involving the measurement of intracellular calcium transits in CHO cells transfected with Cb1 or Cb1+GFP demonstrate that AEA does not evoke the release of intracellular calcium transits from the ER. While we had originally hypothesized that the forced coupling of gng3 to the heterotrimeric pair of G proteins that would associate with Cb1 would result in the ‘switch’ of a Gs like response to a Gq like response, our data fails to corroborate our hypothesis. Similar to the experimental model we used to evaluate a Gs-like response in our cAMP assay, we sought to determine whether forced expression of gng3 could evoke a Gq like response. Several attempts to measure the release of intracellular calcium in cells expressing Cb1+GFP or Cb1+gng3 were made using AEA concentrations of 10uM and 100uM. In all trials, AEA application had no effect on intracellular calcium. Our data is in line with other studies that show Cb1 does not evoke increases in intracellular calcium (Howlett et al., 1987; Howlett et al., 2002). However, other studies using varying cell types refute these findings. For instance, (Hegyi et al.,2018) and Navarrete and araque (2008) demonstrated that application of the Cb1 receptor agonists 60 induced a Gq-like response by increasing levels of intracellular calcium. In an elegant experimental design using primary rat astrocytes, Hegyi et al. (2018) showed that application of Cb1 receptor agonists (10 uM anandamide, 2-AG, and WIN) induced the release of intracellular calcium. These researchers also showed that AEA evoked the strongest calcium response when compared to 2-AG and WIN. This response was absent in Cb1 knock out mice, indicating that the response is occurring through activation of the Cb1 receptor. Moreover, this response was blocked by pretreatment of cells with AM251 (a Cb1-specific antagonist). Although we failed to observe an increase in intracellular calcium evoked by stimulation of AEA, differences in our experimental design may be at fault. Our experiment used AEA to stimulate CHO cells expressing Cb1, while the aforementioned studies used primary astrocyte cell lines. Considering astrocytes are found in the CNS and directly involved in processes mediating neuronal excitability, it is reasonable to assume that these processes would be unlikely to occur in CHO cells (an epithelial-derived cell line). Moreover, these researchers observed differences that were dependent on the Cb1-specific agonist that were used. While all studies did observe calcium increases in response to AEA, it is possible that using a different Cb1 agonist may have evoked increases in intracellular calcium. On the basis of the specific cellular context being pertinent to the response, several studies suggest that Cb1 can act synergistically with other GPCRs, which alters its ability to stimulate G protein pathways. This was demonstrated by Moreno et al.,(2017) who conducted experiments showing that Cb1 is co-expressed in various subpopulations of neurons in the brain (i.e. cortical GABAergic interneurons or Glutamatergic neurons). These studies suggested that Cb1 may act as a G-protein ‘sink’ by sequestering the 61 readily available G-proteins and thus making them unavailable for other receptors. Similar conclusions were also reached by Vasquez and Lewis (1999) using superior Cervical Ganglion (SCG) cells in rats. In the context of our experimental design, Cb1 would not be expressed at the correct stoichiometric proportions with other receptors that are present in neurons under normal physiological conditions. The most notable differences between these studies and ours is their use of primary cell lines compared to our use of CHO-K1 cells. Another possibility that can partially reconcile the apparent Gq-like response evoked by Cb1 stimulation could be that the response does not occur through the actions of the Gq pathway. Offering support to this hypothesis, Daniel et al.,2004 revealed that application of WIN onto parallel fibers of the rat cerebellum inhibited presynaptic calcium transits. This effect persisted when cells were pre-treated with toxins that prevent the activation of Gi/o, N, P/Q and R type Ca2+ channels (PTX, ω-agatoxin TK, ωconotoxin GVIA, SNX-482, respectively). The response was not blocked when treated with tertiapin-Q, a toxin specific to G protein-gated inwardly rectifying K+ channels (GIRKs)—suggesting the actions are mediated by GIRKs. An important difference to note is that our experimental model was unable to determine whether AEA would inhibit calcium transit considering a Ca2+ -free buffering solution was used to prevent interference with the Fura-2AM fluorophores. Arguing against a Gq independent mechanism came later in 2005 when Lauckner et al.,2005 demonstrated that HEK-293 and cultured hippocampal neurons increased intracellular [Ca2+] only when treated with the Cb1 receptor agonist WIN. The response was abolished when pretreated with the Cb1 antagonist SR141716A and acted 62 independently of Gi/o proteins as the response persisted and was enhanced when treated with PTX. It is important to note that this response was WIN Specific and did not occur in cells treated with THC, HU-210, CP55, 2-AG, Methandamide and CBD. Researchers concluded that this response was mediated through the actions of Gq proteins and Phospholipase C (PLC) considering the response was attenuated in cells expressing dominant negative Gq, or when treated with PLC inhibitors. Moreover, this response was blocked by the sarcoplasmic/endoplasmic reticulum Ca2+ pump inhibitors. Considering we were unable to generate any sort of intracellular calcium response mediated by AEA stimulation, the use of these inhibitors would not have been beneficial in the context of our experiment. While there has been a plethora of studies aimed at identifying the specific response mediated by activating Cb1, key differences in experimental design (e.g. cell type, ligand, and experiments conducted in vivo or in vitro) make it challenging to elucidate the underlying mechanisms involved in Cb1 receptor transduction. While these differences are unlikely an intrinsic property unique to the Cb1 receptor, it certainly highlights emerging properties of GPCR’s role in signaling through multiple pathways. 6. Validation of a Visual Motor Response (VMR) Paradigm in Zebrafish Larvae Prior to generating mutant zebrafish, we wanted to validate a behavioral paradigm sensitive enough to detect subtle differences between our mutants and the wild-type phenotypes. We planned to characterize the phenotypes of the mutants with and without the treatment of AEA. The differences between wild-type and mutant phenotypes would be attributed to the mutation, whereas the differences between the phenotype of untreated versus treated mutants would be attributed to the absence of g3 coupling to the Cb1 63 receptor. To be sure that our injections did not have an effect on zebrafish behavior that would be reflected in our data and later attributed to the mutation, we operated off the hypothesis that zebrafish injected with GFP would have a similar VMR when compared to wild type. Our data showed that on average, both WT and GFP mutant zebrafish juveniles travelled \ significantly less in phase 3 (high-intensity light) than those in phase 2 (low-intensity lighting). There are no significant differences between the average distances travelled by GFP mutants in phase 1 or 2. Since we did not calculate the distance travelled in phase 2 of our WT group, we are thereby unable to accept or reject our hypothesis that our injections do not have effect on VMR. Although unable to accept or reject our hypothesis regarding the VMR in GFP compared to WT, our analysis showed that in both phase 1 and phase 3 of our experiments, there are no significant differences in distance traveled between WT and GFP mutants. Therefore, it is reasonable to assume that microinjection itself did not dramatically effect locomotion. Our data is in partial agreement with other similar VMRs conducted using juvenile zebrafish. The relatively large standard errors within groups were attributed to the individual biological variation that is inevitable when using live model organisms (see below). While the obvious outliers may have been omitted from the dataset for ease of determining significant differences, this would have been subjectively determined and would therefore interfere with the integrity of the dataset. Therefore, this variation was a significant factor in determining the significance between groups and may have been reconciled using a larger sample size. Using a similar experimental design, Emran et al.,(2008), demonstrated that on average, zebrafish have distinct responses to sudden changes in light intensity. Zebrafish 64 larvae (4dpf) increased their activity more than double following the transition from high intensity lighting to dark. When the lights are turned back on, activity levels return to basal within a short period of time (~30 seconds). This is in partial agreement with our data showing an increase in activity in the dark phase of GFP mutants followed by activity levels dropping below that of those in dark phase. Several important differences between our study and the study conducted by Emran et al. (2008) may be responsible for the relatively modest increase in activity under dark conditions when compared to those observed by Emran et al.,(2008). For one, our sample size was much smaller which may have prevented our analysis from capturing these dramatic differences in activity. Additionally, Emran et al.,(2008) used fish at 4dpf whereas we utilized fish at 7dpf. When considering the rapid maturation of the zebrafish nervous system in the early stages of development, it is not surprising that the overall movement in zebrafish larvae may be more dramatic compared to those in later stages of development. Research conducted by Burgess and Granto, (2007) using WT zebrafish found that reduction in illumination resulted in a period of transient hyperactivity. Burgess and Granto (2007) found that the increase in activity was characterized by large angle turns that persisted for a period of 5 minutes following reduction in illumination. In comparison to our experimental design, the total distance during the whole period lights on or off was the only parameter measured. We did not characterize the movements or turns in our study, so we are unable to attribute the hyperactivity to any specific types of movement. In hindsight, it would have been beneficial for us to not only measure the distance travelled, but to also characterize specific movement patterns and bending. 65 Similarly, Ganzen et al. (2021) showed a dramatic increase in distance traveled during the dark phase. Although, the increase was only present for a short period of time (~5 seconds) after the transition from light to dark. While Burgess and Granto (2007) and Emran et al. (2008) also reported transient hyperactivity under low illumination, the effect persisted for about 5 minutes and 30 seconds, respectively. Yet again, the duration of hyperactivity between these studies refutes each other. More recently, in what appears to be the most well-controlled VMR study, researchers elegantly demonstrated a linear relationship between the cumulative distance travelled and light intensity between 2kLux and 10kLux, suggesting that stimulus intensity is a direct function of light intensity. These data may explain discrepancies among other studies of similar experimental design (Beppi et al., 2021). Adding more complexity to the existing data on the VMR response in zebrafish, Tuz-sasik et al.,(2022) conducted a series of experiments to evaluate how different experimental illumination settings effected locomotor behavior. In all experiments, zebrafish juveniles were more active under light conditions when compared to dark conditions. While this data refutes data obtained from our study showing that GFP injected juveniles were most active under low light conditions, the researchers only compared locomotion between two conditions, Light and Dark. Our paradigm utilized a Light-Dark-Light set up which revealed that fish were initially more active under high illumination in phase 1 when compared to Phase 3. Importantly, Tuz-sasik et al.,(2022) found that the activity under light was greater when light was presented last compared to when light was presented first. Researchers attributed this phenomenon to acclimation or habituation of the environment. In the context of our data, it is possible that in our study, 66 the fish expended higher amounts of energy when activity levels were relatively greater during phases 1 and 2. Moreover, our fish were under high intensity lighting prior to acclimating to the light box where their arenas were housed. This change from highintensity overhead lighting to high-intensity (but more direct) lighting in the arena may of induced an anxiety-like response during their acclimation, thus partially explaining the relatively higher levels of activity in phase 1 compared to phase 3. Nevertheless, the dramatic differences observed in activity that were dependent upon the order of either light-dark or dark-light make it difficult when comparing the results of our study. Many of these studies differ from ours in several ways including the software used to measure movement, specific behavioral endpoints of interest, duration of light/dark exposure, acclimation periods and the arenas used in assaying. These slight differences in experimental design have minute—yet summative effects on the results obtained and illustrate the complexity of how neurobehavioral paradigms make it difficult to extrapolate biologically relevant findings. Moreover, linking simple changes in locomotion to a specific molecular event is certainly not straight forward and should be recognized as an important limitation to the VMR assay. It Is important to mention several factors that complicated our analysis and that we hope may be of good use for future researchers interested in utilizing a similar experimental paradigm. For instance, ImageJ had difficulty tracking the movement of the juveniles under Low-light conditions. Even after manually adjusting the threshold for individual wells, the software was unable to identify the subtle differences between the juvenile’s zebrafish and the white background that the fish were in. Moreover, the abrupt change in light intensity bleached out the frames following phase 1 and phase 2. This 67 prevented us from identifying the individuals that startled with a “C or O” like bend and from measuring the initial response (distance travelled) following a change in light intensity. While this may be an inherent problem associated with filming under low-light conditions, some simple remedies may offer a solution. Future researchers may be able to utilize two cameras that are situated above the arena. With one camera being adjusted to low light intensity, and the other to high light intensity, the frames could be extracted and matched to the appropriate phase. Another possibility would be to adjust the exposure to a setting that would accurately capture the response in both low and high intensity lighting. This would require a researcher to carefully monitor and appropriately adjust the exposure and light intensity to a level that can be accurately captured, while still eliciting the same light/dark response. Another issue was the depth of the wells, which would inevitably “hide” the zebrafish when they would around the edges. This made it impossible to track the location of the juveniles when they would leave the field of view. Our initial resolution was to lower the amount of water used in each well which would limit movement of fish in the vertical plane, thereby preventing them from moving into the blind spot. However, the amount of water would severely limit their movement and would have made it difficult for future studies where a defined amount of water was needed for treatment with AEA. Perhaps a better solution to this would have been to use the same wells that was utilized in the auditory response trials. Nevertheless, we recommend researchers perform several trials at different camera angles and/or arenas to determine how to best remedy this issue. 7. Validation of an Acoustic Startle Paradigm in Zebrafish Larvae 68 In addition to the VMR, we wanted to validate an assay that could be used to characterize mutant zebrafish. In anticipation to the generation of our mutants lacking the g3 subunit, we tested weather AEA treatment (10uM) would have an effect on the acoustic startle response. We hypothesized that WT juveniles treated with 10uM AEA would have a significantly delayed startle latency and would travel less compared to the untreated control. Our results showed no significant differences between the total distance travelled or the startle latency between treated and untreated juveniles. Moreover, the results obtained in our duplicate trials did not differ significantly from each other. Our results lead us to conclude that AEA has no effect on the acoustically evoked startle response in zebrafish juveniles at 7dpf. To the best of our knowledge, this is the first study to look at the startle response in zebrafish exposed to AEA. However, one major limitation in our research is the lack of a positive control— a test compound known to affect the startle response that would have demonstrated that our assaying system was capable of measuring these differences. Our experiments were performed in duplicate using several fish receiving treatment with AEA and no treatment. While we were sure to include equal amounts of fish of both the treatment and control group in each trial, our experiments were performed in duplicate, thus presenting another inherent limitation. Although an analysis between both trials showed no significant differences were present, suggesting that our experimental setup is unlikely to have significant effect on the any differences that would have been observed from our trials. Our results suggest that Anandamide treatment has no effect startle latency or mean distance traveled in zebrafish, though we are cautious when making this interpretation because of these mentioned limitations. 69 Using a novel method where activity levels are quantified using horizontal and vertical line breaks, Smith et al., (2021) demonstrated that AEA treatment has a dose dependent effect that significantly increases physical activity in adult zebrafish. While this study differs from ours by both the paradigm used to measure activity and the age of zebrafish, they reasoned that expression of Cb1 in the basal ganglia and cerebellum may be responsible for the increase in physical activity. Additionally, they observed that fish in the treatment group were less likely to spend time around the edges and bottom of the arena, suggesting that the treatment may have anxiolytic properties. While it may be tempting to attribute the differences in activity levels in our study compared to the ones obtained by Smith et al., (2021) to a lack a fully developed endocannabinoid system in juveniles, this is unlikely. Oltarbella et al.,(2017) showed that many of the genes that makeup the endocannabinoid system in zebrafish were stably transcribed at 48hpf. Moreover, Migliarini and Carnevali (2009) showed that treatment with the synthetic cannabinoid AM251 dramtically decreased activity levels of in zebrafish larvae as early as 96 hpf. One possibility that may partially explain why no changes in activity were observed in our study, despite the clear support for the endocannabinoid systems involvement in activity found in other studies is the time spent treating and measuring activity in the zebrafish. Smith et al., (2021) treated fish for 30 minutes and Migliarini and Carneveali (2009) chronically exposed larvae to the agonist throughout its development to 96hpf. Moreover, both studies were interested in the how the treatment effected overall activity levels in the fish. Our study is limited in this area considering our treatments with AEA were only for 10 minutes and the overall activity levels were not 70 measured. In hindsight, a longer treatment followed by recording basal activity levels before the startle would have been beneficial. Perhaps another explanation to these differences observed in our studies come from the fundamental mechanism that generates the startle response. The M-cell circuit responsible for eliciting the auditory startle response in zebrafish is mediated by sensory neurons that synapse M-cells responsible for the activation of descending reticulospinal neurons (Xu et al., 2021). We reasoned that because Cb1 receptors are found on reticulospinal neurons in zebrafish (Watson et al., 2008) and an inherent function of these receptors are to inhibit presynaptic neurotransmission, it is likely that activation of these receptors would perturb the ability of these neurons to produce an action potential. Moreover, we reasoned that the effects would be evident in any stimulus that activates this M-cell circuit. However, recent data published during our experimentation by Beppi et al., (2021) showed that the distance travelled during an acoustically evoked auditory startle response was significantly higher when the stimulus was 126 dB compared to all other lower intensity stimuli. This finding corroborates the claim that the intensity of the stimulus has direct effects on the M-cell mediated response and means that the intensity of the stimulus used in our experiment (120dB) likely “overpowered” the inhibition caused by Cb1. In a series of elegantly designed experiments aimed at functionally characterizing the cannabinoid receptors function in relation to locomotor behavior in zebrafish larvae, Lutchenburg et al., (2019) found that treatment with the exogenous cannabinoid receptor agonists WIN55,212-2 and CP55,940 decreased locomotion in larvae at 5dpf. This was observed in both basal, and startle-induced locomotion. Moreover, these effects were 71 mediated by the Cb1 receptor considering the effects were abolished in mutant Cb1 -/fish. Importantly, Lutchenburg et al., (2019) speculated that endocannabinoids at this level of development are not active in regulating locomotion considering that use of the Cannabinoid receptor antagonist AM251 alone has no effect on regulating locomotion. While this certainly corroborates our findings showing no differences when the endocannabinoid AEA was administered, it poses an interesting question— Why do zebrafish possess all relevant components of the endocannabinoid system in the areas known to regulate locomotor activity at this stage of development if they serve no biological purpose in locomotor behavior? This question is especially relevant when considering their findings that activation of the Cb1 receptor effects locomotor activity in zebrafish. It is likely that considering many of the endocannabinoids are produced ondemand (Krug et al., 2015), there were no receptor-bound endocannabinoids available for the antagonists to act on. Nevertheless and in the context of our experiment, the use of synthetic agonists by Lutchenburg et al., (2019) and our use of the endocannabinoid AEA may of likely contributed to the differences in behavior observed. Treatment with several different compounds have been known to modulate acoustic startle responses in developing zebrafish. . For instance, treatment with amorphine was shown to reduce the startle latency. Whereas pretreatment with the D2 receptor antagonist haloperidol enhanced the startle latency. Moreover, Haloperidol was able to attenuate the amorphine induced reduction to startle latency (Burgess and Granato, 2007). Another study conducted by Pantoja et al.,(2016) also showed that amorphine significantly increased the total distance traveled after startle in zebrafish. Interestingly, these researchers also demonstrated that pre-treatment with the serotonergic agonist 72 quipazine alone had no effect on startle, but could also attenuate the startle response of amorphine treated zebrafish (pantoja et al., 2016). These studies demonstrate a significant dose-dependent effect on movement in zebrafish treated with the varying pharmacological agents. This contrasts with our experimental design which only used a single concentration of anandamide (10uM). In hindsight, it would have been beneficial to use multiple concentrations of Anandamide that would allow us to determine if dose had any effect on movements. Moreover, considering the multiple levels of cross-talk occurring between receptors of the endocannabinoid system and the receptors of other systems (i.e. dopaminergic, serotonergic, etc)(Chiang and Chen 2013, Colangeli et al., 2021), co-treatment of AEA with drugs acting on these systems may of revealed novel endocannabinergic interactions that regulate locomotor behavior in zebrafish. M-cell mediated responses have a shorter onset (<12 ms) when compared to non-M cell mediated responses (~28 ms) that also increase swimming velocity (Roberts et al.,2011). Due to the frame-rate of the camera used in our experimental design, we could only use the presence or absence of a C-bend to validate the response as M-cell Mediated. While a C-bend was observed in all subjects where an increase in swimming velocity of 2 standard deviations above the mean swimming velocity were present, future research using a higher frame-rate camera may find it useful in characterizing responses that are not mediated by the M-cell circuit. 8. Design of CRISPR guide RNA Several factors must be considered when designing a guide RNA that will deliver the cas9 endonuclease to the coding region of the proposed gene of interest. These factors 73 include finding an area of the genome that contains the proper Protospacer-adjacent motifs (PAM) sequence that corresponds to the cas9 being used, assuring that the sequence in this area is not found in other sections of the genome, and that there are enough 5’ and 3’ flanking nucleotides for designing the appropriate primers (Sorien, et al.,2018). Considering that the Gng3 transcript is relatively small (232bp) and separated into 3 exons, 2 of which are coding, we chose to target the largest coding exon, (Exon 3, second coding exon) in the zebrafish genome using the UCSC Genome Browser. This sequence was then entered into IDTs genome editing tool and the sgRNA containing the highest on-target and lowest off-target score were chose for our experiment. As will be discussed below in the following sections, this method proved useful in our experiments. Next, we will discuss the methods used in other gene-targeting experiments involving CRISPR/Cas9 in the zebrafish genome, and how they compare to ours. The identification of genomic sequencing suitable for CRISPR targeting would be a daunting task without the use of open-source biotechnology programs. While the details and configurations of these programs are outside the scope of this discussion, these programs can sort through large sets of genomic data and identify relevant PAM sites required for the Cas9 nuclease to cut. The relevant target sites are then compared to sequences within the entire genome to allow researchers to pick the sequence that has the lowest probability of generating off-targets Indels. The work presented by Varshney et al., (2015) uploaded the entire zebrafish genome into a program called Bowtie (first described by Langmead et al., 2009) to identify PAMs that were used to design their guides. Similar to our design using IDT’s genome editing tool, Varshney et al., (2015) used bowtie to identify target regions with the lowest off- 74 target score to avoid generating off-target mutations. This method of sgRNA design allowed them to generate high-efficiency mutations (as discussed below). One drawback to their sgRNA design method was the presence of 5’ mismatched nucleotides, which was reported to have a significant effect on its targeting efficiency. While one of their objectives was to evaluate the high-throughput efficacy of bowtie in designing sgRNA used for gene targeting, it highlighted an important factor for future researchers to consider when designing gRNA. Fortunately, our sgRNA did not contain mismatched bases in the 5’ or 3’ end, as these sets were omitted by IDT. However, due to slight genetic variation among individuals and the possibility of Single Nucleotide Polymorphisms (SNP), it may have been beneficial for us to ensure that we were not targeting a region of the exon known to harbor SNPs, especially near the 5’ end. Similar to the open source programs from IDT and Bowtie, Brocal et al.,(2016) used a program originally designed for use in mouse and human genomes to generate sgRNA (see review by Doench et al., (2016) for review on the CRISPR package). These researchers sought to develop a highly efficient pipeline method of generating and evaluating zebrafish CRISPANTs. Brocal et al., (2016) method relied on the in vitro transcription of the sgRNA which required additional steps to insert the T7 promoter sequence, followed by the guide RNA sequence and the overlap sequence. Our method precludes these additional steps as we utilized IDT to synthesize the crRNA which was assembled to the tracrRNA (which constitutes the sgRNA) immediately prior to injections. Considering that many of the open-source programs are available for sgRNA design are similar in that they use an organisms genetic code to search for unique areas that are 75 amenable to the cas9 technology, it is often based on researchers preference on what programs are utilized. Still, it is important that researchers have a general understanding of how these programs are designing the guides and what “rules” they are using in their determinations. This will allow researchers to use discretion when choosing the appropriate sgRNA design by taking into consideration other factors, like the size of the gene and ability to create suitable 5’ and 3’ flanking primers. Moreover, an understanding of the design logic of the programs used will undoubtedly be beneficial for troubleshooting. While many of the aforementioned studies relied on methods using complicated software and programs, we found that our method in designing sgRNA was simple, user friendly and cost effective. However, it is important to note that these studies were not only targeting different genes but were interested in designing a high-throughput pipeline. 9. Microinjection of zebrafish embryos To successfully target the zebrafish Gng3 gene with our Cas9 endonuclease, we needed to streamline a system that would allow us to obtain multiple WT embryos almost immediately after becoming fertilized while simultaneously preparing and loading the CRISPR components into the microinjector system. We performed several trial runs of embryo rearing to ensure we could successfully breed, capture, and deliver the embryos to the microinjector while they were still in the 1-2 stage of development. The sgRNA was prepared using individual crRNA and tracrRNA components. The RNP complex was assembled using recombinant Cas9 and sgRNA and loaded into the microinjector apparatus with 0.08% phenol red for visualization. Prior to the injection of embryos with 76 the Crispr components, several trial injections were conducted where a GFP solution with 0.08% phenol red was used to assure that the injection process would not be a factor in the developing embryos. Not only did this step serve as a control for the injection process, but it also gave us practice operating the microinjector and visualizing both uninjected and injected embryos in parallel during development. Our results support that the microinjection procedure is safe, and that using recombinant cas9 and chemically synthesized crRNA and tracrRNA to form the sgRNA and RNP complex can be used in the microinjection of zebrafish embryos. Research conducted by Chang et al., (2013) demonstrated the effectiveness of microinjection of CRISPR-Cas9 in zebrafish embryos. These researchers used a cloningbased method where the RNP complex was assembled in vitro from cas9 and the gRNA mRNA that was transcribed from a commercial transcription kit. Cas9 (300ng/uL) and the preassembled gRNA of 20ng/ul into the embryo. Moreover, Chang et al., (2013) used a luciferase recombination assay as a method to screen the embryos early in development for successful delivery and uptake of the CRISPR components by the cells. In brief, this method requires co-injecting a plasmid containing the code for two truncated luciferase fragments. When a double stranded break (DSB) occurred by the cas9/gRNA, the luciferase activity could be measured. While this method would certainly be useful as a means of preliminary screening of F1 mutants, it may result in unwanted side effects from the plasmid being incorporated into the hosts genome Kim et al., 2018). In comparison to our microinjection methods, we assembled the RNP ex-vivo using a 2:1 ration of recombinant cas9 to sgRNA (as described by Sorien et al., 2018). Instead of performing preliminary screening on our mutants using luciferase, we performed mock 77 trials where EGFP was injected into the WT embryos and viewed under fluorescent microscopy. While it may have been a good control to perform these injections in parallel to the CRISPR injections, it would have been very difficult considering the time it takes to load and prepare the microinjector system. Brocal and colleagues (2016) used methods that were similar to Chang et al., (2013). In-vitro assembly of the RNP complex was accomplished by annealing target specific nucleotides containing a T7 promotor sequence to the reverse compliment of the tracrRNA scaffold. The resulting product was then transcribed using a commercially available transcription kit and injected into embryos at a ratio of 20:1 Cas9:sgRNA. While these researchers do not provide any citations or reasoning to this ratio, it is likely that Brocal et al., (2016) wanted to assure that sufficient amounts of cas9 would be available for the RNP complex to quickly form. This is, in part, the reason we chose to assemble to RNP complex ex-vivo (using 2:1 recombinant cas9:sgRNA), as we would not have to worry about the embryo undergoing further development while the cas9 was being translated and assembling into the RNP. Moreover, IVT has been shown to cause innate immune responses in the host immune cells (Mefferd et al., 2015). Similar methods of in-vitro transcription and purification were used to generate the sgRNA template and cas9 transcript by Varshney et al., (2015). An important difference in the experimental design of Varshney et al., (2015) to ours and the other studies mentioned in this section was the goal of targeting multiple genes in each injection or using multiple guides targeting the same gene. This required them to use proportionately more of the cas9 transcript and multiple sgRNAs in their injections (Varshney et al., 2015). While we initially considered targeting multiple regions of Gng3 78 in our injections, we ultimately decided that the relatively short length of Gng3 may have been overcome by large Cas9 proteins attempting to find and cut the target sequence. For embryo rearing prior to injecting, we set up breeding tanks containing 1 adult male and 1 adult female. The female fish that appeared gravid (swollen abdomens) were chose and placed in a breeding tank with a male fish chosen at random. Initially, this method allowed for ~30-40 embryos per tank. However, we noticed large amounts of variability in the total number of embryos produced which became problematic during our injections. Nasiadka and Clark (2012) recommended housing male and female fish in separate tanks until breeding was commenced. We found that after attempting this method, our embryo yields decreased. While we are unable to state with certainty why this common practice for embryo rearing was not successful in our lab, we speculated that the presence of 1 or 2 misidentified males or females in the tank was counterproductive in our study. Many of the aforementioned studies detail methods of the in-vivo assembly of the RNP complex that differ dramatically in terms of concentrations used in the microinjection procedure. This method is not only time-intensive and costly—as it requires multiple steps to synthesize, purify and cap the cas9 mRNA, but also depend on the use of snRNA or tRNA promoters that are constitutively active to drive the in-vivo production of the RNP (Zhang et al., 2017). Other methods utilize the hosts machinery to assemble to RNP by cloning the guide RNA into a plasmid vector containing the Cas9 sequence (Chang et al., 2013). While all of these methods operate off of the same premise—the formation of the RNP by crRNA (containing the sequence complimentary to the target) and tracrRNA (the scaffold portion that binds to cas9)( (see cui et al., 2018 79 for a review of sgRNA and RNP design tools), they have been shown to result in adverse side effects, especially when phenotyping the F1 generation. Here, we describe a method of injecting pre-assembled and solubilized cas9-sgRNA RNP’s. While we cannot preclude the possibility of off-target mutations generated outside the region amplified by our primers, it is reasonable to assume that our method is comparable to the in-vivo assembly considering they do not differ in terms of their fundamental mechanism that ultimately results in the generation of indels. 10. Analysis of zebrafish gDNA for indel formations An array of techniques has been employed by researchers to evaluate the functionality of a protein. These techniques often involve the use of single stranded nucleic acid probes that bind to and prevent the mRNA transcript from being translated into a functional protein. The corresponding phenotype is thus presumed to be a result of the defective or absent protein. While these methods have certainly been successful in elucidating protein function—they are often time sensitive, costly or limited by large amounts of mRNA that out-compete the probes. Our research aimed to utilize a method that is not only costeffective but relies on the permanent deletion of the gene early in the development of the fertilized embryo, thus eliminating the possibility of the DNA template from being transcribed into an mRNA transcript and subsequent translation of the protein product. Here we present the successful use of the CRISPR/Cas9 system to effectively target gng3 in zebrafish. Using the methods of gRNA design and microinjections mentioned above, we were successful at targeting the gng3 gene in developing zebrafish with target 80 efficiency ranging from 12%-95%. Moreover, our study further validates using heteroduplex banding as an initial method for F0 mutant screening of indels formation. While we initially set out to characterize the function of the gng3 gene in zebrafish using the VMR and acoustic stimuli assay, certain factors had prevented this analysis— an experimental error in trial 1 of our microinjections of CRISPR components when attempting to extract the gDNA from the embryos of our first round of CRISPR injections. Additionally, a low yield in embryos produced during our trial 2 of injections prevented us from collecting a negative control of uninjected embryos that would allow for a later comparison of survival rates. Although we did perform a mock injection, trial 0, of embryos that were injected with 0.08% phenol red and eGFP and found no difference in the survival rates of the injected vs uninjected group—suggesting that our injection procedure and the care for embryos following the injections was safe and not a factor in mortality but does not preclude the possibility of an any experimental errors that may be attributed to their deaths (Survival rates in Tables 5-7). The advent of multiplex sequencing and DNA barcoding has been extensively used to detect the formation of Indels in CRISPR targeting experiments (Brocal et al., 2014 and Varshney et al., 2015). In the work presented by Varshney et al., (2015), target efficiency was calculated using multiplex illumina sequencing data from the PCR products of their Crispants. The sequence data was then uploaded into a program they developed and validated called ampliconDIVider (https://research.nhgri.nih.gov/software/amplicondivider/). For our experiments, target efficiency was calculated using the Snythego Interference of CRISPR Edits (ICE) tool 81 (for details and validation of this tool, see Hsiau et al., (2019)). While these programs are both used to quantify the identity and prevalence of genomic edits, the major difference is that the Synthego ICE tool uses sanger sequencing data as an input whereas the ampliconDIVider uses sequence data obtained from Next Generation Sequencing (NGS). We found that the Synthego ICE tool was both user friendly and cost effective by using the sanger sequence data as an input. Nevertheless, Varshney et al., (2015) reported target efficiencies between 75-99% whereas our method ascertained a target efficiency of between 12-93%. An important difference to note is that Varshney et al., (2015) method utilized several sgRNAs directed toward the same gene in their injections, whereas we used a single sgRNA targeted that targeted gng3. In the methods used by Chang et al., (2013), researchers were able to ascertain a target efficiency of ~35%. After isolating DNA from crispants at 50 hpf, the samples were subjected to in-house sanger sequencing. Target efficiency was quantified based on relative band intensity compared to their WT samples. It is important to note that this research had occurred before the development of many of the bioinformatic tools that automated this process. While our study does not offer a direct comparison between Synthego’s ICE tool and the direct visualization of band intensity for generating target efficiency scores, other studies have provided support that this method is not as accurate as statistical-based software (Wu et al., 2015 and Germini et al.,2018) Currently, there are only a handful of well-validated methods used to characterize InDel formation in the genomic sequences of CRISPR gene-targeting experiments. While the technical details of these methods vary, they all require researchers to extract and sequence the genomic DNA from the crispants. The DNA sequences are then compared 82 to those of the WT to obtain relative target efficiency scores that represent the amount of alleles in the sample harboring an insertion or deletion. Similar to the protocol described by Sorien et al.,(2018), we used the appearance of heteroduplex bands or a reduction in homoduplex band intensity to identify possible indel formation in our F0 embryos. The corresponding PCR products were then sent for sanger sequencing and uploaded into Synthego’s ICE analysis tool to determine gene targeting efficiency. 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BioProtocol, 7(4). https://doi.org/10.21769/BIOPROTOC.2148 96 Figure 1: The Endogenous Cannabinoid System 97 Gα Gαs , Gαolf G-protein effector Adenylate cyclase (+) Gαo Gαi1-3 Gαt1,2 , Gαg , Gαz Adenylate cyclase (-), cGMP phosphodiesterase(+) Phospholipase C-b (+), p63RhoGEF P115RhoGEF, PDZ-RhoGEF Inwardly rectifying K+ channels (+) Gαq , Gα11 Gα14 , Gα15/16 Gα12, Gα13 Gbg Gb1g2 (Steiner et al., 2006) Gb5g2, Gg2, Gb1 Gb1, Gg1, Gg2, Gg3, Gg13 (Huang L, et al 1999) Gβ1, Gγ2 (Tabak et al., 2019) Phosphoinositide 3-kinase Gβ1-3g2 (PI3K) Gαo Gαi1-3 , G α13 Voltage dependent Ca2+ Gβ2g2, Gγ3, Gβ1γ3 channels, SNAREs Table 1: Non exhaustive table of G protein subunits and their effector pathways (modified from D.A. Brown, T.S. Sihra, 2008 and Syrovatkina et al., 2016) 98 Figure 2: Activation of a GPCR. 1) Ligand binding : Ligand binds to a receptor in the inactive state at a site within the 7-TM alpha helix bundle resulting in a conformational change to the receptor complex. 2) GDP/GTP exchange: GDP will be exchanged with GTP and the alpha subunit will dissociate from the beta/gamma dimer. 3) Effector Activation: Alpha and Beta will activate multiple and often overlapping signaling cascades and generate second messengers. 4) Signal termination: The signal is terminated by following the hydrolysis of GTP-> GDP by the ATPase domain of the alpha subunit. 99 Level of interaction Cell type Type of interaction Expression of interacting cellspecific substrates Neuron-type Expression-signaling efficacy discrepancy Extracellular Coactivation Intracellular/Subcellular G protein availability Trafficking Compartmentalization Receptor Oligomerization Phenotype/Effect Cannabinoid Receptor interacting protein 1A (CRIP1A) inhibits Cb1 mediated signaling. The presence/absence of Cell specific Adaptor proteins alters Cb1 localization. Cell-type specific isozymes of Adenylyl cyclase can be either inhibited or activated by different Gbg combinations (Raehal and Bohn, 2014; Rozenfeld and Devi, 2008; Rhee et al., 1998) Significant differences in G-protein activation and receptor regulation in hippocampal /cortical GABAergic and Glutamatergic neuron (Steindel et al, 2013) Brain areas with lower levels of Cb1 expression have significantly higher G-protein dependent signaling compared to areas with higher levels of Cb1 (Breivogel et al, 1997) Cb1 receptor-mediated signaling is modulated when coactivated with serotonin or A2A agonists, Attenuated with µ-opioid agonists, and inhibited with GABA B and glutamate antagonists (Garcia et al., 2018) Sequestering of G proteins limiting availability for other receptors (Vasquez and Lewis, 1999) Activated Cb1 can continue signaling during endocytic formation and lysosomal fusion until late endosome stage. Lipophillic CB1 agonists allow Cb1 trafficking to the membrane to become activated before reaching the membrane (Rozenfeld and Devi, 2008; Thibault et al., 2013) Mitochondrial Cb1 receptors (mtCB1) on outer mitochondrial membrane and can directly impact Mitochondrial respiration (Fišar Z, et al. 2014) Cb1 receptor Heterodimerization with D2 (Marcellino D , 2008) , µ-opioid, Orexin, and A2a receptors results in the convergence of signaling pathways leading to attenuation, potentiation or modulation of Cannabinoid signaling Several endogenous and exogenous Cb1 agonists stimulate distinct G protein heterotrimers (Diezalarcia, R, 2016) Biased agonism Table 2: Non-exhaustive list of the multiple levels of signaling pleiotropy in the Cb1 receptor 100 Figure 3: Map of the pcDNA3.1 restriction sites and inserts. 101 Figure 4: Map of the pcDNA3.0 containing EGFP and restriction sites 102 Figure 5: Schematic showing the sgRNA sequence and a schematic of the CRISPR/Cas9 system targeting the second coding exon (exon 3) in Gng3. 103 Primer Sequence 5’à3’ Annealing Cycles temperature (Celsius) 68 30 Size (bp) 253 AP03 AP04 TGACTTCCTTCAGGGGTAGT TCCAGAACTGTGAAGGTGCC WS93 WS94 ACGCAAGATGGTGGAACAG GGGCATCACAGTAAGTCATCAG 68 30 95 WS99 WS100 TTCGCCGCCATGTCTTC GTCACAGTAAAGCACAGGATCT 64 35 217 AP07 AP08 AP11 AP12 Target use Cnr1 for C. griseus and golden hamster gng3 in C. griseus and golden hamster Gng10 in C. griseus and Golden hamster ATCTCTTTCTTGATGTCTCCTGTAT 64 35 183 gng3 in zebrafish TAGACTAATCCTGGGCGTCCT AGAAATGGACTCTTTTGCGTTCA 65 35 314 gng3 in zebrafish TGGTGTCTCGTCGAGTGTTG Table 3: PCR primers. PCR reactions were carried out with an initial denaturing temperature of 95C for 1 minute, and a subsequent denaturing temperature of 95C for 30 seconds at the start of each cycle. Annealing for 1:30 seconds at indicated temperature. Extension was carried at 70C for 3 minutes. 104 Figure 6: Embryos lined up on a glass side in a petri dish in preparation of microinjection 105 Figure 7: PCR products of cDNA isolated from Hamster Brain (Brain) and CHO-K1 (CHO) cells amplified using Crn1 (AP03-AP04), Gng3 (WS93-WS94) and Gng10 (WS99-WS100) specific primers. Visualized on a 1.5% agarose gel stained with Ethidium Bromide. Panel A; Lane 1: Brain, band at 253bp (Cnr1). Lane 2: CHO, no band present (Cnr1). Lane 3: Negative control, no band present (Cnr1). Lane 4: Brain, band at 95bp (Gng3). Lane 5: CHO, no band present (Gng3). Lane 6: Negative control. No band present (Gng3). Lane 7: 100bp ladder. Panel B; Lane 1: Brain, band at 217bp (Gng10). Lane 2: CHO, band at 217bp (Gng10). Lane 3: Negative control, no band present (Gng10). 106 Figure 8: Restriction Enzyme Digest of plasmids visualized on an agarose gel verifying the identity of the plasmid:Lane 1- 1KB DNA ladder , Lane 2- gng3 undigested (band present ~5660), Lane 3- gng3 (XhoI and HindIII digest) (Band present ~ 5408 and 250bp), Lane 4-Crn1 Undigested (band at 6846), Lane 5- Crn1 (XhoI and EcoR1 digest) (band at 5427 and 1419), Lane 6- EGFP undigested (band at 6159), Lane 7- EGFP (Xmn1 digest) bands at 3381 and 2778. 107 Figure 9: Standard curve showing the linear relationship between cAMP concentration and ∆RLU values (n=3) using the cAMP-Glo assay. 108 Figure 10: FURA-2AM Intracellular calcium tracings in CHO-K1 cells expressing Cb1+Gng3 (Panel A), Cb1 (Panel B) and CB1+GFP (Panel C) treated with 10µM or 100µM AEA (indicated by dashed line). Tracings indicate no change in the levels of intracellular calcium upon treatment with 10µM or 100 µM AEA. 109 250 Distance (mm) 200 150 100 50 0 Phase 1 (GFP) Phase 1 (WT) Phase 2 (GFP) Phase 3 (GFP) Phase 3 (WT) Figure 11: Average distances travelled during the VMR assay in WT and GFP injected zebrafish. Phase 1 consisted of 4 minutes of high intensity lighting following 10 minutes of acclimatation. Phase 2 consisted of 4 minutes of low intensity lighting (dark challenge). Phase 3 consisted of 8 minutes of high intensity lighting. On average, fish in both groups travelled less in phase 3 than in phase 2, however, there are no significant differences between treatment groups. 110 14 12 10 8 6 4 2 23.7 22.8 21.9 21 20.1 19.2 18.3 17.4 16.5 15.6 14.7 13.8 12 Control 12.9 11.1 9.3 10.2 8.4 7.5 6.6 5.7 4.8 3.9 3 2.1 1.2 0.3 0 Treatment Figure 12: Distance travelled before and after sound blast (120dB) in WT zebrafish. Zebrafish were treated with 10uM of AEA for 15 minutes before recording started. Blast occurred at 0.33 seconds. The average distance travelled between treatment and control are insignificant. Dotted line indicated blast of horn. 111 Trial 1 Trial 1 before Trial 1 after after Trial 2 Trial 1 before blast blast blast before blast (treatment) blast (control) (treatment) (control) (treatment) Average (mm) stderr 3.25 0.07 3.15 0.10 54.84 1.15 57.75 1.39 2.60 0.84 Trial 2 before Trial 2 after Trial 2 after blast blast blast (control) (treatment) (control) 3.55 0.15 Table 4: Average distance travelled in Trials 1 and 2 of Startle Response. 112 35.59 0.08 43.15 1.24 Seconds until turn Figure 13: Histogram showing the distribution of average startle latency travelled in Control (Panel A) and Treatment (Panel B) groups. 113 Figure 14: PCR products of gDNA isolated from Crispants. DNA was amplified using primers specific to gng3 flanking the cut site (AP11-12) and ran on 1.5% agarose gel stained with ethidium bromide (Lane 1: 2-1, Lane 2: 1-6, Lane 3: 2-5, Lane 4: 2-6, Lane 5: WT, Lane 6: water blanks, Lane 7: 100bp ladder). Banding is present in lanes 1-5 at ~314bp corresponding to the expected wild-type sequence in lane 5. Heteroduplex banding (lanes 2 3 and 4) indicates possible Indel formation. 114 Figure 15: Indel plot from samples 2-1 (panel A), 2-5 (panel B), and 2-6 (panel C), showing the Indel percentage (percentage of the pool with non-wild type sequence; Xaxis) versus indel size (change in base pair between non-wild type and wild type sequences; y-axis). The R2 value is from a linear regression generated by fitting inferred editing outcomes with observed editing outcomes and indicates the confidence level of the ICE score. 115 Figure 16: Photo of sample 2-5 prior to extracting gDNA (panel A) and chromograms of the edited sample (panel B) and Wild type sample (panel C). The small peaks under the full sequence traces in panel B represent the non-edited gDNA. The dotted vertical lines in panels B and C represent the cut-site and the horizontal black line in panel C indicate the guide sequence. 116 Injection Trial 0: EGFP + Vehicle injected Uninjected (n=51) Injected (n=18) 24hpf survival 61% 69% 48hpf survival 100% 100% 72hpf – 7dpf survival 100% 100% Table 5: Survival rates for Injection Trial 0. Zebrafish embryos were collected and microinjected with a solution containing an EGFP + Nuclease-Free Duplex Buffer. Survival rates for both injected and uninjected embryos was determined. 117 Trial 2- CRISPR Injection 24hpf survival 76%, n=10 48hpf survival 100% 72hpf survival 100% 96hpf survival 0% Table 6: Survival rates for Injection Trial 1. Zebrafish embryos were collected and microinjected with the CRISPR solution. Survival rates for both injected and uninjected embryos was determined. 118 Trial 2- CRISPR Injection 24hpf survival 76%, n=10 48hpf survival 100% 72hpf survival 100% 96hpf survival 0% Table 7: Survival rates for Injection Trial 2. All Zebrafish embryos were collected and microinjected with the CRISPR solution. 119 Appendix 1. Animal Research (IACUC) approval information and Reports Date: October 12, 2020 To: Dr. William Schwindinger, Alex Pascule From: Dr. Candice M. Klingerman, IACUC Chair Re: IACUC Approval of Research Protocol Your protocol for the project referenced below has been approved by the Institutional Animal Care and Use Committee for the period of time specified in the application. Please keep in mind that if you plan any significant changes to your animal procedures during the time period covered by this protocol you must receive IACUC approval before they are implemented. If you are unsure whether a proposed change is a significant one that requires IACUC approval, feel free to contact me with questions. Title of Project: Pleiotropic Signaling in the Endocannabinoid System: The role of the γ subunit Protocol number: 172 Approval Period: Fall 2020, Spring 2021, Summer 2021 cc. Dr. R. Lynn Hummel, Interim Dean of College of Science and Technology Sadie Hauck, Director of Research and Sponsored Programs 120 TO: FROM: Bloomsburg University Faculty Performing Research with Animals Alex Pasculle DATE: August 18, 2020 RE: New Research Protocol Form and Guidelines The Institutional Animal Care and Use Committee (IACUC) recently approved a revision of the IACUC protocol form. This new protocol must be complete before research with nonhuman vertebrate animals can be performed at Bloomsburg University. This form must be submitted and approved prior to nonhuman vertebrate animals use in: 1) classroom demonstration/experimentation 2) experimental research 3) naturalistic observation Forms can be typed using a word document template on the S:drive, print six (6) copies and submit to Chairperson. If this protocol has been previously approved fill out Section A only and submit six (6) copies of the previously approved protocol and acceptance letter. If you have any questions, feel free to call me at 4953 or e-mail at cshonis@bloomu.edu. Bloomsburg University Bloomsburg, Pennsylvania Animal Research Protocol Form Section A (must complete): Protocol # (Chair will assign) Instructions: This form should be completed and six (6) copies sent to the Chairperson of the IACUC. The review will be completed within two (2) weeks. Protocols must by TYPED. Students must have the protocol co-signed by their faculty advisor. Projects involving experimentation or naturalistic observation require protocols. Name of Investigator(s): Alex Pasculle, William Schwindinger Department: Biological and Allied Health Sciences 121 Title of Project: Pleiotropic Signaling in the Endocannabinoid System: The role of the γ subunit Semesters in which animals will be used (check all that apply and include year): Fall ____√____ Year _2020_____ Spring __√_____ Year _2021_____ Summer __√____ Year _2021_____ Species of Animals: Zebrafish (Danio rerio) Approximate number of animals being used: 65 adult male and female zebrafish and 235 zebrafish embryos. Has this protocol been previously approved? No If yes, give protocol # and attach a copy of the approved protocol along with the letter of approval. If the present protocol is a replication of the previous one then it is not necessary to complete the rest of this form. Simply sign this form and submit it with a copy of the previously approved protocol and acceptance letter (six (6) copies of everything). Section B (fill out only if new protocol): What type of hypnotics (i.e. sedatives, analgesics, anesthetics) will be used to eliminate pain sensation if surgical procedures will be performed? N/A If no hypnotics will be used to eliminate pain sensation in surgery, give complete rationale: No anesthetics will be used to eliminate pain because the early stage zebrafish embryos (3-7dpf) do not feel pain or distress (Matthews et al. 2012) and will therefore be unharmed during the CRISPR injections. What euthanasia method will be used at the end of the experiment? Euthanasia will be accomplished by rapid chilling in 2-4°C degrees for 10 minutes (AVMA Guidelines on Euthanasia: 2020 Edition). Adult zebrafish will be euthanized with tricaine methane sulfonate (TMS). Euthanasia will be performed by immersing the fish in 200 mg of TMS in 1 liter of water and sodium bicarbonate to buffer the solution to a pH of 6-7 until they are no longer moving or breathing. They will then be rapidly decapitated (Harper and Lawrence, 2011). Present a brief rationale for involving animals, and the appropriateness of the species and numbers to be used. • Cannabinoid receptor signaling in vertebrates and invertebrates are conserved (Elphick and Egertova 2001) 122 • • • • • • • • The CB1 and CB2 receptor have 99% and 88% Amino-acid sequence homology between humans and zebrafish, respectively (Klee 2012) In both humans and zebrafish, Cb1 and Cb2 expression is conserved and localized to the same structures, with high Cb1 expression in the CNS , specifically the telencephalon, hypothalamus, tegmentum and anterior hindbrain and peripheral Cb2 expression (lam et al. 2006) Expression of the Cb1 receptor transcript in zebrafish begins at 24 hpf (lam et al 2006) allowing for a quick turnaround time to assay after injection Moreover, gng3 is conserved among humans and zebrafish and share 93.3% sequence homology (NCBI BLAST) Heterotrimeric G-proteins function as important mediators in signal transduction in both humans and zebrafish. Zebrafish represent a good animal model because their development occurs externally, and their embryos are transparent. Moreover, high fertility, small size and relatively sort generational time will allow high-throughput phenotypic characterization. It is important to use animals in vivo to study behavior, as in vitro or other methods are inappropriate. We will use appropriate numbers of living of adult zebrafish (min n=65, max n=75) in this study to minimize the number of fish used while achieving adequate numbers for statistical analysis. Zebrafish can give rise to several hundred embryos each week, which will allow us to limit the amount of zebrafish being utilized for generating embryos. The number of embryos to be used in these experiments are similar to those of Lutchenburg et al. (2019). To the best of your knowledge, does this project duplicate an activity (e.g. research or classroom demonstration) that you or others have conducted: __yes______. If yes, give scientific rationale for duplication. A project similar to experiment 2 has been already been conducted and approved by IACUC. In the previous project, line crosses were used to characterize zebrafish activity after exposure to low and high dose anadamide. The experiment will be repeated in order to better quantitate zebrafish activity using IDtracker software. The assays will then be used at a later date when full homozygous gng3 knockouts are obtained. Experiment 0 Trial using uninjected Wild Type zebrafish embryos to validate a startle response assay Summary Startle response assays have been well-characterized and are routinely used to evaluate neurological, behavioral and motor function in developing zebrafish (Colwill et al., 2011). We have chosen experiments that measure startle response early in development (tactile, visual and auditory)(figure 1). The main purpose of this experiment is to modify and optimize the startle response assay (using uninjected WT embryos) that will be used in experiment 1b. 123 Methods Startle response protocols: A video camera mounted above a petri dish will record the response evoked when the embryos are stimulated (Either by an acoustic, tactile or light stimuli). The videos will be analyzed using open-source software that will quantify locomotive behavior available at www.opencv.org (information on the software available here): The direction of the Cbend, the time it takes until the tail touches the head and the time spent active after dark/light challenges. A Chi-square test will be used to compare responsive vs nonresponsive zebrafish. Tactile Startle Response (3dpf) The assay will be conducted in two phases. Phase 1 will consist of acclimating the zebrafish larvae to a quiet and light room for 5-10 minutes. Phase 2 will consist of inducing a tactile stimulus to the larvae and recording the response. The petri dish will be placed on a vortex prior to assaying. At the start, the vortex will be turned on low for a brief period of time (about 1 second). Visual startle response (4dpf) (adapted from MacPhail et al., 2008) The assay will be conducted in two phases. Phase 1 will consist of acclimating the zebrafish larvae to a period of darkness in the 96-well plate for 5-10 minutes. Phase 2 will be a light challenge where lights will be turned on for 10 minutes and recording will begin. The activity of the larvae will be compared. Auditory startle response (5dpf) (adapted from Zeddies et al., 2005) The assay will be conducted in two phases. Phase 1 will consist of acclimating the zebrafish larvae to a quiet room in a 96-well plate for 5-10 minutes. Phase 2 will begin after an acoustic stimulus is sounded above the recording apparatus. The activity of the larvae following each acoustic stimulus and time required for startle response initiation will be analyzed and compared between groups. Acoustic stimuli will be delivered using a prerecorded sound played through free-field speakers placed 1-2 feet away from the experimental set up. Experiment 1 total animals: *The same Wild-type zebrafish from experiment 1 will be used to rear embryos. 1. WT embryos (N=10) Total embryos= 10 ! Figure 1- Time course the startle response assays 124 Experiment 1. In vivo Knock down of gng3 using a sgRNP CRISPR-Cas9 Summary It has been well established that endocannabinoid signaling mediated by the Cb1 receptor is pleiotropic (Lutchenburg et al. 2019). While much focus has been on the Gα and Gβ subunits, the objective of this experiment is to determine the role of the Gγ3 subunit in activation of the Cb1 receptor. To determine the functional relevance of the Gγ3 subunit in zebrafish, we will first generate gng3 (Gγ3 subunit) knockout mutants using CRISPR/Cas9 injected into F0 embryos. This method is preferred over other knockout approaches in zebrafish have often been complicated by factors like the partial duplication of the zebrafish genome, and the fact that other methods of genomic engineering may only be effective early in development (Cornett et al., 2018). Methods Fis h Ca re Fish will be brought into the freshwater fish room (Hartline Science Center; room B55), group-housed (~30 fish per 10 L aquaria (up to 100 fish in each is appropriate; Harper and Lawrence, 2011), and allowed to acclimate to the facility for at least 7 days. Afterwards, they will be separated into individual (or small-grouped), 3 L holding tanks (See Figure 2). 10 L 3L Figure 2. zebrafish aquaria Fish will be placed on a 14:10 day/night light cycle to induce spawning. Water temperature will be maintained at 28°C/82°F with aquarium heaters. An air pump will deliver oxygen to the water. Water will be filtered through a reverse osmosis/ deionized (RO/DI) filtration system (Spectrapure) and delivered automatically to each aquaria from a holding tank. Water conditioner (Aqueon) and Instant Ocean 125 Sea Salt (0.5-2.0 g/L) will be added. Aquaria are specially-made to fit inside of a holding rack (Aquatic Habitats Benchtop System; Pentair Aquatic Ecosystems). See Figure 3 for a similar set up. A filter is located below the holding rack. The filter will contain material for biological, chemical, and mechanical filtration. Figure 3. zebrafish aquaria setup Initially, water quality will be checked daily using an API Freshwater Master Test Kit. After water quality becomes stable, some parameters, like nitrogen, can be tested weekly. Oxygen will be tested weekly using an oxygen sensor. Water pH will be maintained between 7-8, alkalinity between 50-150 mg/L CaCO3, hardness at least 75 mg/L CaCO3, salinity between 0.5-2 g/L, dissolved oxygen at 2 mg/L, carbon dioxide below 20 mg/L, and nitrogenous waste less than 0.02 mg/L (Harper and Lawrence, 2011). The objective in Experiment 1 is to generate gng3 KO zebrafish to use for behavioral testing in other experiments. Microinjection Protocol (Adapted from Rosen et al., 2009, Sorlien et al., 2018) Crosses will be set up the night prior to F0 embryo collection by placing fish in a divider breeding tank. The dividers will be lifted, and the fertilized embryos will be collected 1 hour after dividers have been lifted. The fertilized embryos will be transferred to a 10 cm petri dish containing water obtained from the same system under the same conditions as the system. • Prior to embryo injection, the sgRNA and CRISPR/Cas9 will be kept frozen and thawed on ice until ready for injection. • Two sgRNAs targeting gng3 at different loci will be simultaneously injected into the F0 embryo and raised until adulthood. Two groups of zebrafish will be generated, a treatment group (gng3-/-) and a negative control group (water injected into embryo). 126 • • • • • • Injections will occur between the hours of 10:00 am and 2:00 pm and will be performed Microinjection solution consist of a 2:1 ratio of Cas9:sgRNA (Invitrogen). The final concentration will be 200pg/nL sgRNA and 400 pg/nL Cas9. The total volume of solution will be 5uL. The petri dish containing the embryos will be inspected under a dissection microscope at 2.5X magnification prior to injection An injection needle will be made by pulling a 1.0mm glass capillary and will be cut at an angle with a razor to ensure that the opening can pierce the Chorion and Yolk sac. The injection will be conducted in an agarose container (Chapter 5 in zebrafish book) The needle will be place in the micromanipulator and attached to a microinjector with the air source turned on. 1 nL of the solution will be injected into the yolk sac of each embryo. Injected embryos will then be transferred to an incubator for development. Experiment 1b- Behavioral Analysis of F0 embryos Summary Individual G-protein subunits have been shown to have specific roles during embryogenesis— including angiogenesis, cell migration and motility. Moreover, Gprotein dependent signaling occurs during development through many different GPCR’s (Syrovatkina et al., 2017). The next step is to determine the phenotype of Gng3/- zebrafish. Startle response assays are used to evaluate neurological and motor function in zebrafish embryos starting as early as 36 hpf and consist of a small stimulus (e.g. light, water-flow, light) applied to the developing embryo (Colwill et al., 2011) This assay was chosen because it has been validated in many studies as a useful model to evaluate various parameters in developing zebrafish that translate well to humans. Hypothesis Gng3-/- zebrafish will have an altered startle response when compared to wild type. Methods Embryos reared in petri dishes from both treatment and control groups will be collected and placed in a 96-well plate containing water from the same system. When a small stimulus is applied to the embryo, the zebrafish will coil up in the opposite direction until the tail touches the head. This is known as the startle response or C-bend and is a behavior that promotes the sympathetic fight or flight response (Colwill et al., 2011). Additionally, other methods of stimulation (i.e. visual, acoustic, tactile) can be performed at various stages of development to characterize escape and avoidance behaviors. This will allow us to obtain a better repertoire of phenotypes exhibited by the zebrafish larvae throughout development. Zebrafish larvae will hatch from their chorion around 2 dpf. We will start by conducting a tactile startle response assay on zebrafish larvae at 3 dpf. This assay will consist a tactile stimulus (waterflow or vibrational stimuli). A Visual startle response assay will be conducted at 4dpf, the developmental period where zebrafish are 127 beginning to develop a visual system. Finally, at 5dpf an auditory startle response will be measured. Zebrafish will be treated with 10 nM anandamide for 2 hpf prior to assaying. These assays were chosen because they are simple, high-throughput, easily quantifiable and will allow for robust screening of larvae early in development. An appropriate number of embryos will be retained following euthanasia for genotype analysis (see figure 4). Experiment 0 outlines the trial experiment that we will use to optimize and validate our experimental design. While we report the protocols for all three assays, experiment 1b will only use one startle response assay (best characterized in experiment 0). . ! Startle response protocols: In experiments using Anadamide, embryos will be treated with10nM for 2 hours prior to assaying (Migliarini et al., 2008). A video camera mounted above a petri dish will record the response evoked when the embryos are stimulated (Either by an acoustic, tactile or light stimuli as described above in experiment 0).The videos will be analyzed using opensource software that will quantify locomotive behavior available at www.opencv.org (information on the software available here): The direction of the Cbend, the time it takes until the tail touches the head and the time spent active after dark/light challenges. A Chisquare test will be used to compare responsive vs nonresponsive zebrafish. Experiment 1 total animals: 1. Wild-type female zebrafish (n=10) 2. Wild-type male zebrafish (n=10) Total Fish= 20 zebrafish 3. Gng3 -/- embryos (n=75) 4. Water control embryos (n=75) 5. Wild type embryos (n=75) Total embryos= 225 128 Experiment 2 To characterize behavioral related phenotypes associated with Cb1 receptor activation in adult Zebrafish (Danio rerio). Summary of Experiment 2 Anandamide is the Fatty acid Neurotransmitter involved in endocannabinoid signaling and has affinity to both Cb1 and Cb2 in zebrafish and humans (Sulcova et al., 1998). Both Cb1 and Cb2 are GPCR’s and are therefore dependent upon the heterotrimeric Gprotein complex. Many studies have previously identified changes in phenotype associated with the loss of an individual G protein subunit (Schwindinger et al., 2004 and Leung et al., 2006). In order to gain a complete understanding of the changes in endocannabinoid signaling associated with an individual G-protein subunit, it is imperative to develop an assay sensitive enough to discriminate between the differences in phenotypes. In this current study, adult zebrafish will be administered a low or high dose of anandamide, and their activity will be measured. This experiment will be used to validate the assay (using IDtracker) and characterize behavioral related phenotypes in Wild-type zebrafish. The data obtained from this experiment will be used later when identifying the phenotypes involved in Gng3 knock out zebrafish. Moreover, it will allow for a better understanding of how endocannabinoid signaling effects the behavioral phenotype of zebrafish. Hypothesis 2 Anandamide will increase fish swimming behavior compared to fish treated with vehicle. Methods Fish housing, and the experimental set-up will be the same as in Experiment 1. After acclimated to the laboratory, fish will be exposed to either 10 uM (low dose) or 100 uM (high dose) of ANA, or vehicle. Anandamide will be purchased as Arachidonoyl Ethanolamide from Sigma Aldrich (A0580) or Cayman Chemicals (90050). The vehicle will be a very small dose of ethanol which will be added to the treatment water at a similar amount as the ANA-treated fish. These doses of ANA have been adapted from Piccinetti et al., 2010. Fish will be exposed to the ANA or vehicle by placing them into a separate treatment tank for up to 1 hour. They will then be placed back into their normal aquaria and physical activity will be recorded using a video camera for an additional hour. Physical activity data will then be analyzed using the software idTracker, http://www.idtracker.es/. All fish will be euthanized after testing. At the time of euthanasia, blood may be collected for additional analysis. Experiment 2 animals 1. Vehicle control with water (n=15) 2. 10um ANA (low dose) (n=15) 3. 100um ANA (high dose) (n=15) Experiment 2: 129 Total number of animals: = 45 130 References AVMA releases new euthanasia guidelines(2020). . Monmouth Junction: MultiMedia Healthcare Inc. Retrieved from Agricultural Science Database database. Retrieved from https://search.proquest.com/docview/ 2377679456 Colwill, R. M., & Creton, R. (2011). Imaging escape and avoidance behavior in zebrafish larvae. Reviews in the Neurosciences, 22(1), 63-73. doi:10.1515/RNS.2011.008 [doi] Corley-Smith, G. E., Lim, C. J., & Brandhorst, B. P. (1995). 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C., & Robishaw, J. D. (2009). Mice lacking the G protein gamma3-subunit show resistance to opioids and diet induced obesity. American Journal of Physiology.Regulatory, Integrative and Comparative Physiology, 297(5), 1494. doi:10.1152/ajpregu. 00308.2009 [doi] Sorlien, E. L., Witucki, M. A., & Ogas, J. (2018). Efficient production and identification of CRISPR/Cas9generated gene knockouts in the model system danio rerio. Journal of Visualized Experiments : JoVE, (138). doi(138), 10.3791/56969. doi:10.3791/56969 [doi] Sulcova, E., Mechoulam, R., & Fride, E. (1998). Biphasic effects of anandamide. Pharmacology, Biochemistry, and Behavior, 59(2), 347-352. doi:S0091-3057(97)00422-X [pii] 131 Syrovatkina, V., Alegre, K. O., Dey, R., & Huang, X. Y. (2016). Regulation, signaling, and physiological functions of G-proteins. Journal of Molecular Biology, 428(19), 3850-3868. doi:10.1016/j.jmb. 2016.08.002 [doi] Zeddies, D. G., & Fay, R. R. (2005). Development of the acoustically evoked behavioral response in zebrafish to pure tones. The Journal of Experimental Biology, 208(Pt 7), 1363-1372. doi:208/7/1363 [pii] I hereby certify that the information contained herein is true and correct to the best of my knowledge. _________________________________________________ ___9/30/20 Investigators(s) Date _________________________________________________ _________________9/30/20 Faculty Advisor (if applicable) Date 132 Appendix 2. Supplemental Figures Supplemental Figure 1: Schematic outlining components of the Fura-2AM experiment 133 Supplemental Figure 2: Schematic showing the experimental work-flow of project. 134