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.

Nitrous oxide (N2O) is a widely used anesthetic adjunct in dentistry and medicine that is also commonly abused. N2O alters the function of several receptors in vitro and ex vivo, however, the receptors systems underlying its abuse-related intoxicating effects are poorly understood. The goals of this dissertation were to (1) establish N2O as a discriminative stimulus, (2) characterize the temporal properties of the discriminative stimulus, (3) determine the degree of similarity between N2O and other inhalants and (4) explore the neurochemical effects responsible for the stimulus properties of N2O. Twenty-four mice were trained to discriminate 10 minutes exposure to 60% N2O+40% O2 from 100% O2 in daily 5 minute food-reinforced operant sessions. Mice acquired the discrimination in a mean of 38 sessions. N2O produced concentration-dependent full substitution for itself. Full substitution required 7 minutes of N2O exposure but the offset of stimulus effects following cessation of N2O exposure were more rapid. Varying degrees of partial substitution for N2O were engendered by abused vapors and vapor anesthetics. The aromatic hydrocarbon toluene produced the most robust substitution for N2O. One or more toluene concentrations produced full substitution for N2O in 7 of 8 subjects, suggesting that these two abused inhalants share common neurochemical mechanisms. The NMDA receptor open channel blockers (+)-MK-801, ketamine and memantine produced dose-dependent partial substitution for N2O. A competitive NMDA antagonist and NMDA glycine site antagonist did not substitute for N2O. Pretreatment with (+)-MK-801 as well as ethanol produced dose-dependent leftward shifts in the N2O concentration effect curve further suggesting some overlap in their mechanisms of action. GABAA agonists and positive allosteric modulators, opioid agonists, serotonergic agonists, nicotine, a nNOS inhibitor and the psychomotor stimulant amphetamine all failed to appreciably substitute for N2O and/or failed to alter the N2O concentration effect curve when administered prior to N2O exposure. No drug tested produced greater than 80% mean N2O-lever selection leaving open the possibility of other neurochemical contributors to the stimulus effects of N2O.

Emerging evidence suggests that opioid drugs can exacerbate neuroAIDS. Microglia are the principal neuroimmune effectors thought to be responsible for neuron damage in HIV-infected individuals, and evidence suggests that drugs acting via opioid receptors in microglia aggravate the neuropathophysiological effects of HIV. The P2X family of ATP activated ligand-gated ion channels regulates key aspects of microglial function. In addition, opioid-dependent microglial activation has been reported to be mediated through P2X4 signaling, prompting us to investigate P2X receptors contribution to the neurotoxic effects of HIV and morphine. In vitro experiments showed treatment with TNP-ATP prevented the neurotoxic effects of morphine and/or HIV Tat, or ATP alone in a concentration dependent manner. This evidence suggests P2X receptors mediate the neurotoxic effects of these insults in striatal neurons. P2X1, P2X3, and P2X7 selective receptor antagonists did not prevent Tat- and/or morphine-induced neurotoxicity, implying cellular pathways activated may not involve these subtypes. Cells from P2X4KO mice show that activation of the P2X4 receptor on glia are necessary to cause Tat and/or morphine toxicity. However, data implied that baseline neuronal function may be altered due to lack of P2X4 receptor expression, and also gave evidence for altered Tat and morphine cellular signaling when the two are given in combination versus alone. Surgeries were performed on P2X4 KO and WT mice, which received intrastriatal Tat injections and morphine and/or naltrexone pellets. WT mice showed significant increases in inflammatory markers when treated with Tat and/or morphine. Increases in inflammatory markers were not seen in P2X4 KO mice, implying P2X4 receptors play a role in neuroinflammation resulting from Tat and/or morphine. Finally, human tissue samples from the National NeuroAIDS Tissue Consortium were analyzed. Changes in P2X5 and P2X7 mRNA were found in microarray data, but only changes in P2X7 mRNA levels were confirmed by RT-PCR. No changes in P2X4 mRNA levels were detected. Our experiments indicate the P2X receptor family contributes to Tat- and morphine- related neuronal injury, and reveal that members of the P2X receptor family, especially P2X4, may be novel therapeutic targets for restricting the synaptodendritic injury and neurodegeneration that accompany neuroAIDS and opiate abuse.

The primary treatment options for head and neck cancer are radiation therapy or surgery, or both combined; chemotherapy is often used as an additional, or adjuvant, treatment. Patients treated with radiotherapy are exposed to a high cumulative dose of radiation over a period of time and there is a 17-33% chance of recurrence. High cumulative doses of radiation, a long time course of treatment, side effects and the possibility of recurrence provide the rationale for developing approaches for radiation sensitization, which could be helpful to patients in decreasing the dose, duration of radiation, side effects, or the chance of recurrence. Radiation induces autophagy, which is a catabolic process involving the degradation of the cell’s own components to generate energy under conditions of stress. Autophagy can be cytoprotective helping the cell to survive during stress such as nutrient deprivation or it can be cytotoxic, leading the cell toward death. We investigated whether blocking autophagy by the use of the antimalarial drug, chloroquine, could sensitize head and neck cancer cells to radiation. Studies were performed using the HN30 human head and neck cancer line (p53 wild type) derived from the pharynx as well as HN6 human cells (p53 mutant) derived from the base of the tongue. Cell viability was determined by cell counting and clonogenic survival assays, autophagy was monitored based on acridine orange staining accompanied by flow cytometry, while western blotting, DAPI and TUNEL staining and PI/annexin/FACS were utilized for determination and quantification of apoptosis. Senescence was monitored by beta-galactosidase staining/ FACS analysis. Radiation alone produced a transient growth arrest followed by proliferative recovery in both the HN30 and HN6 cancer cells. Radiation also promoted autophagy in both cell lines. The combination of chloroquine with radiation inhibited autophagy and promoted apoptotic cell death and suppression of proliferative recovery for the HN30 cells, but had little effect on sensitivity to radiation and proliferative recovery in the HN6 cells. The data suggest that autophagy induced by radiation serves a protective function in the HN30 cells and that a blockade to autophagy by chloroquine drives the cell toward apoptosis and death. In contrast, autophagy in HN6 cells appears to be non-protective as a pharmacological blockade did not sensitize the HN6 cells to radiation. These studies support the premise that autophagy induction by radiation need not necessarily have a cytoprotective function and further indicates that caution should be exercised in efforts to sensitize head and neck cancer to radiation through the clinical suppression of autophagy.

An individual’s naïve level of response (LR) to ethanol is predictive of their lifetime likelihood to abuse alcohol. LR is heavily genetically influenced, suggesting that the genes responsible for LR may also be central to the development of abuse disorders. Our laboratory uses the model organism C. elegans to investigate the genetic influences on responses to acute ethanol exposure. We recently found that changes in TAG levels can alter LR. From this result we investigated the role of long-chain polyunsaturated fatty acids (LC-PUFAs) as well enzymes involved in lipid modifications of proteins. We found that LC-PUFAs are necessary for acute functional tolerance and that supplementation of eicosapentaenoic acid is able to rescue AFT. We also identified mutations in several palmitoyltransferases, a thioesterase, and elongases that alter AFT. These novel results highlight the importance of fatty acids in the response to ethanol and suggest exciting new potential therapeutic targets.

There is significant sequence homology and anatomical co-distribution between cannabinoid (CB1) and sphingosine-1-phosphate (S1P) receptors in the CNS, but potential functional relationships between these lysolipid receptors have not been examined. Therefore, to investigate possible relationships between these two systems at the level of G-protein activation, agonist-stimulated [35S]GTPγS binding and autoradiography were conducted. Autoradiographic studies were first performed to localize receptor-mediated G-protein activation in mouse brain. Coronal brain slices were processed for stimulation of [35S]GTPγS binding using the synthetic cannabinoid agonist WIN 55,212-2 (WIN) or SIP. High levels of WIN- and S1P-stimulated [35S]GTPγS binding were observed in the caudate putamen, hippocampus, substantia nigra, and cerebellum. To further characterize the relationship between S1P-and CB1-mediated G-protein activation, spinal cords from adult male CB1 receptor knockout mice, CNS-deleted S1Pl receptor knockout mice and wild type C57 mice were collected, and assessed using agonist-stimulated [35S]GTPγS binding. Results from this experiment revealed that the S1Pl receptor is predominant in mouse spinal cord. To further investigate potential CBl and SIP receptor interactions spinal cords were collected from adult male ICR mice. Additivity studies were preformed using agonist-stimulated [35S]GTPγs binding. Results showed significantly less than additive stimulation when spinal cord tissue was treated with both WIN and SIP. These results suggest an interaction between the CB1 and S1P receptors in the mouse spinal cord. The effect of cannabinoid antagonists, SR141716A (CB1) and SR144528 (CB2) on S1P-and WIN-stimulated [35S]GTPγS binding were also examined in mouse spinal cord homogenates. These results showed that there was no significant difference between S1P-stimulated [35S]GTPγS binding in the presence of SR141716A or SR144528 compared to vehicle control. This shows that S1P produced stimulation independent of the CBl or CB2receptor. In addition WIN-stimulated [35S]GTPγS binding was not affected by SR144528, but was inhibited by SR141716A, confirming that this action is due to the CB1 receptor. The combined results of this project demonstrate an interaction between CB1 and S1P receptors in certain CNS regions where they are co-distributed, such as the caudate putamen, hippocampus, substantia nigra, cerebellum and spinal cord. These results may be due to convergence on a common pool of G-proteins via dimerization or co-localization in lipid rafts, or a possible direct ligand-receptor interaction.

In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D3, also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D3 also enhanced radiosensitivity and promoted autophagy in MCF7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D3 failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D3 was not attenuated by either a pharmacological or genetic block to autophagy; this was due largely to the promotion of apoptosis via the suppression of protective autophagy that occurs in response to radiation alone. Moreover, pharmacological blockade of autophagy did not sensitize noncancerous MCF10a cells to radiation; conversely, 4T1 mouse mammary tumors were highly sensitive to pharmacological inhibition of autophagy, suggesting selective radiosensitization against cancer cell lines. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D3). In addition, this work highlights the technical challenge of establishing the potential cytotoxic function of autophagy in an experimental system where the cytoprotective function may be concurrently expressed.

Tobacco dependence is high in women who suffer from anxiety disorders yet little is known about the contributions of nicotinic acetylcholine receptors (nAChRs) on anxiety-like behavior. β2*nAChRs (*denotes assembly with other subunits) are the most abundantly expressed nAChRs in the brain yet little is known about the contributions of β2*nAChRs on anxiety-like behavior in female mice. In this study, antagonism and nicotine effects on anxiety-like behavior was investigated across the life span in 6, 12 and 24-month-old drug-naïve knockout (KO), heterozygous (HET) and a gain of function α6L9S mice and wild type (WT). HET mice showed increased sensitivity to di-hydrobeta-erythroidine compared to WT mice. Aged mice showed decreased locomotor activity and exploratory behavior compared to younger mice. Low doses of nicotine produced anxiolytic-like effects, whilst a high dose of nicotine produced anxiogenic-like effects. Activation of the α6*nAChRs supports an anxiolysis-like phenotype. These results implicate α4β2*nAChRs and α6β2*nAChRs in anxiety-like behavior.

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.

Numerous findings have suggested that the increased prevalence of childhood illnesses such as cancer, infection, and allergy may be due to environmental exposures. One such cause may be maternal smoking or passive smoke exposure. Known immunotoxicants in cigarette smoke and environmental pollution include polycyclic aromatic hydrocarbons such as 1,2:5,6 Dibenzanthracene (DBA). The objective of these studies was to evaluate the immunosuppressive effects of DBA on various stages of immune system development. Adult mice were administered DBA daily in corn oil at dose levels of 158, 500, 1580, and 5000 µglkg S.C. for 28 days. Immunosuppression was not observed at levels less than 5000 µgkg in the following immune parameters: NK cell activity, anti-CD3 antibody-mediated proliferation and mixed-leukocyte response. In contrast, holistic assays such as the PFC response to the T-dependent antigen, sRBC and the delayed type hypersensitivity response were significantly suppressed at dose levels of 500 µglkg and greater. Mice exposed to DBA in utero and through lactation showed neither immunosuppressive nor sex differences among the immune parameters tested when evaluated at weaning, postnatal day (PND) 21, or when evaluated at sexual maturity (PND 42). Transference of DBA metabolites from mother to pup is suggested by HPLC analysis of milk extracted from PND 8 pups. In contrast, juvenile mice administered DBA beginning on PND 21 at dose levels from 0.25 to 2500 µgkg for 28 days demonstrated a dose-dependent suppression (43-79%) of the PFC assay, statistically significant at or above the 2.5 µglkg dose level. Neither immunosuppressive nor sex differences were observed among the various other immune parameters evaluated. Collectively, these studies indicate that the juvenile life stage in B6C3F1 mice is the most vulnerable to DBA-induced immunotoxicity with a 200-fold enhancement in immunosuppression of the PFC response as compared to adult mice. These studies provide insight into how environmental contaminants, such as DBA, may impact children's health.