While there is currently active debate over possible therapeutic applications of marijuana and cannabis-based compounds, consistently their primary drawbacks have been the psychoactive properties, dependence, and abuse potential. Prolonged administration of ∆9-tetrahydrocannabinol (THC), the primary psychoactive constituent in marijuana, demonstrates both tolerance and physical withdrawal in both preclinical and clinical studies. Repeated THC administration also produces CB1 receptor adaptations in the form of reduced activation of receptors, along with a downregulation of membrane surface receptors, in many brain regions involved in THC-associated behaviors. The increased need for drug to maintain therapeutic effects, and a withdrawal syndrome following discontinuation of use, are common risk factors in drugs of abuse. Recently, compounds have been developed that prolong the availability of the major naturally occurring endogenous cannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), through inhibition of their catabolic breakdown by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The overall objectives of this research are to elucidate the physiologic roles of these two endogenous ligands and to determine if either can produce beneficial therapeutic effects without negative cannabis-like CNS effects. Therefore, we tested the impact of acute and prolonged blockade of FAAH and MAGL on a variety of cannabinoid-mediated behaviors and on precipitated cannabinoid withdrawal. Despite that acute blockade of FAAH and MAGL produce similar efficacy in reducing nociceptive responses, and both can reduce THC-induced somatic withdrawal, sustained blockade of these enzymes leads to remarkably different adaptations in CB1 receptor functioning. Namely, prolonged elevations in brain 2-AG leads to marked antinociceptive tolerance, cross-tolerance to exogenous cannabinoid agonists, and physical dependence. In contrast, sustained elevations in brain anandamide continues to dampen pain responses without apparent signs of physical withdrawal, loss of CB1 receptor activation as measured by [35S]GTPγS, or receptor downregulation as measured by [3H]CP,55940. These results suggest that chronic 2-AG elicits greater compensatory changes in CB1 receptor functions than anandamide. With similar efficacy in most therapeutic endpoints tested, and evidence of reduced impact on long-term function of the endocannabinoid system, these results distinguish FAAH as a more promising therapeutic target to treat pain and other conditions than MAGL.

CB1 receptors (CB1R) mediate the psychoactive and therapeutic effects of cannabinoids including ∆9-tetrahydrocannabinol (THC), the main psychoactive constituent in marijuana. However, therapeutic use is limited by side effects and tolerance and dependence with chronic administration. Tolerance to cannabinoid-mediated effects is associated with CB1R adaptations, including desensitization (receptor-G-protein uncoupling) and downregulation (receptor degradation). The objectives of this thesis are to investigate the regional-specificity in CB1R function and regulation. Previous studies have investigated CB1Rs in a subset of regions involved in cannabinoid effects, but an inclusive regional comparison of the relative efficacies of different classes of cannabinoids to activate G-proteins has not been conducted. A novel unbiased whole-brain analysis was developed based on Statistical Parametric Mapping (SPM) for 3D-reconstructed mouse brain images derived from agonist-stimulated [35S]GTPgS autoradiography, which has not been described before. SPM demonstrated regional differences in the relative efficacies of cannabinoid agonists methanandamide (M-AEA), CP55,940 (CP), and WIN55,212-2 (WIN) in mouse brains. To assess potential contribution of novel sites, CB1R knockout (KO) mice were used. SPM analysis revealed that WIN, but not CP or M-AEA, stimulated [35S]GTPgS binding in regions that partially overlapped with the expression of CB1Rs. We then examined the role of the regulatory protein Beta-arrestin-2 (βarr2) in CB1R adaptations to chronic THC treatment. Deletion of βarr2 reduced CB1R desensitization/downregulation in the cerebellum, caudal periaqueductal gray (PAG), and spinal cord. However in hippocampus, amygdala and rostral PAG, similar desensitization was present in both genotypes. Interestingly, enhanced desensitization was found in the hypothalamus and cortex in βarr2 KO animals. Intra-regional differences in the magnitude of desensitization were noted in the caudal hippocampus, where βarr2 KO animals exhibited greater desensitization compared to WT. Regional differences in βarr2-mediated CB1R adaptation were associated with differential effects on tolerance, where THC-mediated antinociception, but not catalepsy or hypothermia, was attenuated in βarr2 KO mice. Overall, studies using SPM revealed intra- and inter-regional specificity in the function and regulation of CB1Rs and underscores an advantage of using a whole-brain unbiased approach. Understanding the regulation of CB1R signaling within different anatomical contexts represents an important fundamental prerequisite in the therapeutic exploitation of the cannabinoid system.

The effects of cannabinoids in reducing somatic signs of opioid withdrawal have been known for some time. In morphine dependent rodents, opioid withdrawal following precipitation with the mu opioid antagonist naloxone elicits robust withdrawal behaviors including jumps, paw flutters, head shakes, diarrhea and weight loss. Delta-9-tetrahydrocannabinol has been shown to reduce this opioid withdrawal in mice via activation of the cannabinoid type-1 (CB1) receptor and recently it has been shown that inhibition of the catabolic enzymes for endocannabinoids also reduce somatic signs of opioid withdrawal. Specifically, inhibition the enzyme fatty acid amide hydrolase (FAAH), the catabolic enzyme for the endocannabinoid N-arachidonoylethanolamide (AEA; anandamide) or inhibition of the enzyme monoacylglycerol lipase (MAGL), the catabolic enzyme for the endocannabinoid 2-arachindonoylglycerol (2-AG) has been shown to reduce opioid withdrawal in mice. However, FAAH inhibition only reduced a subset of withdrawal signs in mice and full MAGL inhibition which maximally reduced somatic withdrawal signs has been shown to produce THC-like effects and dependence potential. Additionally, the effects of endocannabinoid catabolic inhibitors on other aspects of withdrawal, such as the negative motivational effects, are not known. The objectives of this dissertation were to 1) assess the efficacy of dual inhibition of FAAH and MAGL on somatic signs of opioid withdrawal and 2) determine whether these treatments would produce cannabimimetic effects (hypomotility, catalepsy, antinociception and hypothermia); 3) develop other behavioral assays of opioid withdrawal; and 4) determine if endocannabinoid catabolic inhibitors would reduce the acquisition of opioid withdrawal induced conditioned place avoidance (CPA) as a measure of the negative motivational consequences of opioid withdrawal. We found that full inhibition of FAAH with the selective inhibitor PF-3845 and partial inhibition of MAGL with the selective inhibitor JZL184 reduced withdrawal-related jumps and the expression of diarrhea to a greater degree than either inhibitor alone and these effects were shown to be CB1 mediated. Additionally, we tested the novel dual FAAH/MAGL inhibitor SA-57 which has greater potency at inhibiting FAAH over MAGL and found that it similarly reduced withdrawal signs at doses that only partially elevated 2-AG while fully elevating AEA; furthermore, SA-57 did not produce cannabimimetic effects at these doses. We next assessed the effects of morphine withdrawal in five behavioral assays: marble burying, novelty-induced hypophagia, the light/dark box, a novel procedure developed to assess “escape behavior” and the CPA procedure. From these studies we selected the CPA procedure to further evaluate the effects of endocannabinoid catabolic inhibitors to determine their ability to reduce the negative motivational aspect of opioid withdrawal. We found that naloxone (0.056 mg/kg) produced robust CPA in morphine-pelleted, but not placebo-pelleted, mice and that this dose elicited minimal somatic withdrawal signs. Morphine pretreatment was shown to block withdrawal CPA and withdrawal jumping in mice while clonidine only blocked withdrawal CPA and these served as positive controls. We found that THC, JZL184, and SA-57 significantly reduced the percentage of mice that jumped during the conditioning session, demonstrating that these treatments blocked the somatic signs of withdrawal. However, none of these treatments significantly affected acquisition of the withdrawal CPA. These studies suggest that dual inhibition of FAAH/MAGL has enhanced effects on attenuating withdrawal-related jumps and diarrhea, but not the negative motivational aspects of morphine withdrawal as inferred by the Pavlovian CPA experiments.

Chronic neuropathic pain is often difficult to treat due to its resistance to therapeutic intervention. This is due in part to the poor understanding of the physiological mechanisms involved in the establishment and maintenance of neuropathic pain states. The neuropathic pain model, chronic constriction injury of the sciatic nerve, produced robust pain hypersensitivity in our mice. It also induced significant changes in the mitogen activated protein kinase family, and the cannabinoid and µ-opioid systems in three different brain areas involved in the modulation or regulation of pain states. CCI induced a 2.5 fold increase in mRNA of the kinase Raf-1 in the PAG of mice. Raf-1 is part of the ERK cascade in the MAP kinase family of proteins. The MAPK family of proteins has previously been shown to be involved in the establishment and maintenance of chronic neuropathic pain via central sensitization and the PAG is a critical regulator of nociceptive input and is part of the descending pain pathway, which has also been shown to have a role in central sensitization. CCI also resulted in significant decreases in the µ-opioid receptor agonist DAMGO stimulated [35S] GTPγS binding in the medial thalamus, and the cannabinoid receptor agonist, WIN 55, 212-2 stimulated [35S] GTPγS binding in the anterior cingulate cortex. These effects were not due to an overall decrease in µ-opioid receptor or cannabinoid receptor 1 binding, suggesting that the chronic pain-like condition resulted in a desensitization of these receptors. Both the medial thalamus and the anterior cingulate cortex are brain areas involved in the medial pain pathway which, along with the limbic system, have been shown to be involved in the affective component of pain processing. These data are the first to demonstrate changes in these three cellular systems in the respective brain areas of the mouse in response to chronic neuropathic pain. The novel findings presented in this dissertation provide new areas of investigation for the treatment of this debilitating disease.

The developmental psychopharmacology of cannabinoids is poorly understood and little is known about the developmental consequences of repeated exposure to cannabinoid antagonists. In these experiments, male Long-Evans rats were treated with SR141716A, a cannabinoid antagonist, throughout adolescence and allowed unrestricted access to food. Control groups were treated with vehicle during the same developmental period and allowed either unrestricted access to food or were pair-fed with a member of the SR-treated group. Motivation to work for food was measured in progressive-ratio sessions at varying levels of food deprivation. For rats that consumed fewer calories throughout adolescence, whether because of pharmacological intervention or food-restriction, motivation was not significantly related to the level of food deprivation. Additionally, the SR-treated group ate more of a novel, palatable food than the vehicle-treated group. Finally, the SR-treated group was generally more motivated to work for food than the pair-fed group, irrespective of the level of deprivation.

Cannabinoids, such as the marijuana derivative Δ9-THC, are known to have CBl receptor-mediated anticonvulsant effects in several animal models of seizures and epilepsy, including the rat pilocarpine model of acquired epilepsy. However, the distribution of CBl receptor expression and function in brains of epileptic rats has not been characterized. Therefore, this dissertation was initiated to evaluate the effect of epileptogenesis on the distribution and function of the endogenous CBI receptor system in the rat pilocarpine model, a well-established model of acquired temporal lobe epilepsy. Using immunohistochemistry, we demonstrated that chronically epileptic rats exhibit a unique, long-term, and specific redistribution of hippocampal CBl receptors when compared to controls, with concurrent layer-specific increases and decreases in CBl receptor expression within the hippocampus. In addition, studies in this dissertation demonstrated using [3H] WIN55,212-2 autoradiography and agonist-stimulated [35S]GTPγS autoradiography that this CBl receptor-specific reorganization results in corresponding functional changes manifested by alterations in CBl receptor binding and G-protein activation. These regionally selective changes were dependent on NMDA receptor activation during the initial insult of pilocarpine-induced status epilepticus (SE), and were independent of seizure suppression produced with phenobarbital administration in epileptic rats. Furthermore, time-course studies utilizing these techniques demonstrate that within a week following SE, a widespread loss of CBl receptor expression and function occurs throughout the hippocampus. The subsequent redistribution of CBl receptors that occurs temporally correlates with the emergence of spontaneous recurrent seizures, and is still observed up to 1 year following SE. Overall, the reorganization of cannabinoid receptors in epilepsy implicates the endocannabinoid system in modulating neuroexcitability in the epileptic state. This CBl receptor redistribution represents an essentially permanent neuronal plasticity change associated with epileptogenesis, and could account for the anticonvulsant effect of cannabinoids observed in this model.

Cysteine cathepsins B, L and S are endosomal/lysosomal proteases that participate in numerous physiological systems. Cathepsin expression and activity are altered during various inflammatory diseases, including rheumatoid arthritis, atherosclerosis, neurodegenerative diseases and cancers. Early immune responses to microbial pathogens are mediated by pattern-recognition receptors, including Toll-like receptors (TLR). Signaling through TLR causes cell activation and release of inflammatory mediators, which can contribute to the severity of chronic inflammatory diseases. The impact of TLR cell activation on cathepsins B, L and S activities was investigated using live-cell enzymatic assays. Individual ligands of TLR4, TLR2 and TLR3 increased intracellular activities of the three cathepsins indicating the involvement of both MyD88-dependent and -independent pathways. To investigate the role of inflammatory cytokines in regulating these proteases, a lipopolysaccharide (LPS) non-responsive cell line was utilized. LPS non-responsive cells co-cultured with LPS responsive macrophages upregulated cathepsin activities. Furthermore, culture supernatants from LPS-stimulated macrophages increased cathepsin activities in LPS non-responsive cells, which could be reduced by neutralizing antibodies to TNF-α or IL-1β. These findings indicate cytokines regulate cathepsin activities during macrophage responses to TLR stimulation. Using LPS as a model for inflammation, the ability of the cannabinoids, delta9-tetrahydrocannabinol (THC), and CP55940 to suppress cysteine cathepsins during an inflammatory response was investigated. Cannabinoids, including the major psychoactive component of marijuana THC, modulate a variety of immune responses and have been proposed as possible therapeutics to control chronic inflammation. Cannabinoids may mediate their effects through receptor-dependent or independent mechanisms. Cannabinoid receptor subtype 1 (CB1) and receptor subtype 2 (CB2) have differential expression in leukocytes. Dose response studies showed that 1 nM THC was sufficient to inhibit cathepsin enhancement in LPS-stimulated cells. P388D1 macrophages expressed CB2 mRNA, but had no detectable CB1 mRNA indicating a role for the CB2 receptor. Utilizing a CB2-/- macrophage cell line, the role of CB2 receptor participation in THC inhibition of cysteine cathepsin upregulation was explored. THC did not affect cathepsin activity in LPS-stimulated cells lacking CB2 expression. These findings support the possibility of receptor selective agonists as therapeutic treatment during inflammatory diseases to prevent cathepsin involvement in pathological tissue destruction.

The idea that humans and laboratory animals can become physically dependent on marijuana or its primary psychoactive constituent, delta-9-tetrahydrocannabinol (THC), is gaining acceptance. However, there are no currently approved pharmacotherapies to treat cannabinoid withdrawal. The objective of this thesis was to evaluate whether elevating endogenous anandamide levels using mice lacking fatty acid amide hydrolase (FAAH), the enzyme responsible for anandamide metabolism, would ameliorate THC dependence. Mice were treated subchronically with a low or high THC dosing regimen and challenged with the CB1 receptor antagonist, rimonabant, to precipitate withdrawal. Following subchronic THC treatment, rimonabant precipitated a significant increase in paw flutters that was dependent on THC dose. However, FAAH-/- mice displayed a similar magnitude of withdrawal responses as wild type control mice, regardless of subchronic dosing regimen. Finally, rimonabant was equipotent in precipitating withdrawal responses in both genotypes. Collectively, these results demonstrate that FAAH-/- and +/+ mice show identical THC dependence, thus arguing against the notion that elevating anandamide levels through FAAH suppression will reduce cannabinoid withdrawal.

Morphine and Δ9-THC are drugs that produce analgesia and rewarding effects. However, chronic treatment with morphine and Δ9-THC produces problematic side-effects including tolerance and physical dependence. The cellular mechanisms underlying opioid and cannabinoid antinociceptive tolerance have been studied for years. Research has demonstrated that the expression of morphine and Δ9-THC antinociceptive tolerance may be mediated through intracellular signaling pathways, such as the adenylyl cyclase /Protein Kinase A (PKA) cascade. The present study investigated the role of PKA in the expression of morphine and Δ9-THC antinociceptive tolerance. Male Swiss Webster mice were treated chronically with morphine or Δ9-THC and the warm-water tail-flick test was used to assess antinociception. These studies revealed that the level and the duration of morphine antinociceptive tolerance both influenced whether PKA activity was increased in mouse brain and spinal cord. Cytosolic PKA activity was increased in the thalamus of 3-day morphine-tolerant mice expressing a 45-fold level of tolerance, but not in mice that expressed a 10-fold level of tolerance. In addition, cytosolic PKA activity was increased in the lumbar spinal cord (LSC) of 15-day morphine-tolerant mice. However, chronic treatment with A9-THC had no effect on neuronal PKA activity even in mice that expressed a high level of antinociceptive tolerance. The absence of an effect of chronic treatment with A9-THC on neuronal PKA activity was supported by the development of a positive control in which the PKA activator Sp-8-Br-cAMPS was administered intracerebroventricularly (i.c.v.) and intrathecally (i.t.) in drug-naYve mice and increases in PKA activity were observed in several brain regions and LSC. Finally, the i.c.v. injection of two peptide fragments of native Protein Kinase A inhibitor (PKI) peptide, PKI-(6-22)-amide and PKI-(Myr- 14-22)- amide, significantly reversed antinociceptive tolerance in mice treated chronically with morphine. PKI-(6-22)-amide (i.c.v.) also inhibited PKA activity in brain regions (thalamus, periaqueductal gray (PAG), and medulla) and LSC, which studies have shown play a role in morphine-induced analgesia. Moreover, PKI-(6-22)-amide reduced the increase in PKA activity in thalamus and LSC observed with chronic morphine treatment. Overall, these studies provide evidence that PKA plays a role in morphine tolerance, but not Δ9-THC tolerance at the doses and times tested.

Cannabinoids produce antinociception in many preclinical models of acute and chronic pain. In contrast, cannabinoids produce inconsistent analgesia in humans, showing little or no efficacy in treating acute pain, with modest efficacy in treating chronic inflammatory pain. This discrepancy may reflect an overreliance on preclinical assays of pain-stimulated behaviors, defined as behaviors that increase in rate or intensity following delivery of a noxious stimulus. In these assays, antinociception is indicated by a reduction in pain-stimulated behaviors, and antinociception is produced either by a reduction in sensory sensitivity to the noxious stimulus (i.e. true analgesia) or by false positive motor impairment. This dissertation addresses this weakness by complementing cannabinoid effects in conventional assays of pain-stimulated behavior with their effects in novel assays of pain-depressed behavior. Pain-depressed behaviors are defined as behaviors that decrease in rate or intensity following presentation of a noxious stimulus. Motor impairment does not produce false positive antinociception in assays of pain-depressed behavior, because antinociception is indicated by a blockade or reversal of pain-induced behavioral depression. In this dissertation, an intraperitoneal (IP) injection of lactic acid served as an acute noxious stimulus to stimulate stretching (pain-stimulated behavior) or depress intracranial self-stimulation (ICSS) (pain-depressed behavior), whereas, IP injection(s) of lipopolysaccharide (LPS) served as a chronic/acute inflammatory-related noxious stimulus to stimulate mechanical allodynia (pain-stimulated behavior) or depress ICSS (pain-depressed behavior). Cannabinoids tested in the assays of acid-stimulated stretching and acid-depressed ICSS included: mixed CB1R/CB2R agonists THC and CP55940, drugs that modulate levels of the endogenous cannabinoid agonist anandamide (URB597 and PF3845), and a selective CB2R agonist, GW405833. THC was also tested in assays of LPS-stimulated mechanical allodynia and LPS-depressed ICSS. In general, mixed CB1R/CB2R agonists were ineffective or exacerbated pain-depressed behavior regardless of noxious stimulus. Contrastingly, URB597 and GW405833 produced antinociception in the assay of acid-depressed ICSS; however their effects were not mediated by CBRs. All compounds produced antinociception in the assay of pain-stimulated behavior, except for PF3845. These results suggest that assays of pain-depressed behavior may be useful for development of cannabinoid analgesic medications, but that further research is needed to determine mechanisms underlying cannabinoid-mediated antinociception in these assays.