The primary emphasis of this dissertation project was to study the pathogenesis of neuropathic pain, and to develop new treatment strategies targeting primary sensory neurons of the pain pathway. Chronic neuropathic pain is debilitating and is maladaptive. Current treatment options are moderately effective with serious side-effects. Translational progress of novel drugs is impeded by side-effects, which is partly due to reliance on inaccurate methods of pain evaluation in animals. Our first step was to develop a novel behavioral assay for quantitation of spontaneous pain in an animal model. Results from this study showed that rats with trigeminal neuropathic pain had reduced drinking efficiency in an orofacial operant assay, whereas control rats maintained normal levels throughout testing.

The second step of this project was to investigate novel peripherally restricted cannabinoids (PRCBs) for the effective treatment of neuropathic pain. Our initial testing in a rat model of sciatic nerve entrapment (SNE) revealed that, systemic administration of a PRCB (0.3 mg/kg) completely suppressed signs of mechanical allodynia between two and three hours. Based on this information, we conducted a preliminary pharmacokinetic study that showed peak plasma drug levels at 2 hrs after administration with negligible traces of drug in the brain and cerebro-spinal fluid (CSF). Subsequently, we tested these novel PRCBs for CNS side-effects due to central activation of cannabinoid receptor subtype 1 (CB1R). Systemic and oral administrations at therapeutic and higher doses showed complete lack of CNS side-effects. These side-effects prevailed after systemic administration of centrally acting cannabinoid, HU-210 in the same group of animals. Site specific administrations and further evaluation in orofacial operant assay confirmed that anti-allodynic actions of PRCB are mainly mediated at the periphery. Our in vitro data showed that PRCBs are full agonists at CB1R and partial agonists at CB2R. Co-administration of PRCB (1 mg/kg i.g.) with receptor specific antagonists revealed that anti-allodynic actions are largely mediated by CB1R. Since tolerance is of main concern with repeated use of chronic pain medications, we tested the anti-allodynic efficacy of PRCB during repeated oral administrations (1 mg/kg, i.g.) and found no appreciable tolerance after two weeks of testing. Western blot analysis showed stable levels of CB1R in the L4-L5 dorsal root ganglia (DRG) of these animals after repeated testing.

The last research aim of this project was to investigate the axonal translocation of Nav1.8 mRNA after peripheral nerve injury. The hyperexcitability of afferent nociceptors is induced by axonal accumulation of certain ion channels and the role of Nav1.8 sodium channel is highly implicated. Therefore, we investigated the axonal translocation of Nav1.8 mRNA. Our qPCR data showed abnormal accumulation of Nav1.8 mRNA along with Nav1.6 and Nav1.9 mRNAs in

the injured infraorbital nerve. Since the 3’ untranslated region (UTR) is essential for sub-cellular localization of mRNAs, we did cloning and sequencing of Nav1.8 mRNA 3’ UTR. Results showed previously annotated short 3’ UTR and an alternative long UTR that is produced as a result of alternative splicing and alternative polyadenylation.

The current research work demonstrated the use of orofacial operant assay for the evaluation of ongoing or spontaneous pain in a rat model of trigeminal neuropathic pain. In addition, our extensive pre-clinical testing of PRCBs for the evaluation of therapeutic efficacy, may aid in the development of these novel drugs as future therapeutics. In conjunction, the newly identified 3’ UTR variant of Nav1.8 mRNA may represent the pathological basis for abnormal axonal accumulation of Nav1.8 mRNA after peripheral nerve injury.

Major anxiety disorders, such as post-traumatic stress disorder (PTSD), have a devastating social impact. PTSD can lead to high rates of substance use disorders, such as alcoholism. Previous investigations show strong interplay between stress and alcohol use, but have yet to characterize precise brain mechanisms involved. Evidence points to the basolateral amygdala (BLA) as playing a central role in the acquisition and storage of emotional memory.

Recently, an animal model of stress enhanced fear learning (SEFL) that mimics specific characteristics of PTSD has been developed to study behavioral and physiological components of PTSD-like stress in rodents. In PTSD, enhanced fear after a traumatic experience is manifested as an inappropriately exaggerated response to stimuli that are reminders of the trauma, as well as an increased propensity to acquire new fears. In SEFL, enhanced fear after an initial stressor is manifest as a sensitized fear response and enhanced fear learning after exposure to subsequent mild stressors. This work shows that the SEFL model leads to increased voluntary alcohol consumption in stressed rats compared to unstressed rats. Interestingly, however, SEFL does not alter previously acquired alcohol drinking behavior.

Biochemical analysis of BLA tissue and electrophysiological recordings in pyramidal neurons from the lateral nucleus of the BLA were done to identify the cellular mechanisms by which SEFL induces enhanced freezing and increased alcohol intake. This work shows that SEFL increased BLA expression of the α-amino-3-hydroxy-5-methy-4-isoxazole propionic acid receptor (AMPAR) subunit, GluA1, and the γ-aminobutyric acid type-A receptor (GABAAR) subunits α1, α2, and α3. SEFL also produced long-term increases in AMPAR-mediated excitatory postsynaptic currents and changes in voltage-sensitive inward rectifying depolarizing currents that regulate the excitability of pyramidal neurons. Together these data demonstrate long-lasting biochemical and functional changes in the intrinsic and synaptic excitability of the BLA pyramidal neurons. Since the BLA is necessary for the induction and expression of SEFL, it is likely that these biochemical and functional changes represent the brain mechanisms responsible for the behavioral manifestations of SEFL.

Migraine headache affects ~15% of the world’s population, greatly diminishing sufferers’ quality of life. Injection of glyceryl trinitrate (GTN), a known migraine trigger in humans, produces hypersensitivity symptoms of allodynia and hyperalgesia in a previously developed mouse model of migraine headache. We developed synthetic non-psychoactive peripherally-restricted cannabinoids (PRCBs) and demonstrated their effectiveness in relieving cancer and neuropathic pain symptoms. Here, we investigated the efficacy of one of our novel PRCBs, PrNMI, in preventing the symptoms of GTN-induced hypersensitivity in C57BL/6J female mice. Von Frey filaments were used to assess GTN (10 mg/kg, i.p.)-induced changes in head or hindpaw withdrawal thresholds. Selective cannabinoid 1 receptor (CB1R) and CB2R antagonists were used to examine receptor subtype contribution to PrNMI’s effects. Following behavioral assessments, collected trigeminal ganglia (TG) were analyzed for various protein biomarkers of TG neuron sensitization utilizing an automated capillary electrophoresis system. Separately, whole-cell patch clamp recordings from acutely isolated TG neurons assessed changes in the activation of low pH- and allyl isothiocyanate-induced currents. Results showed that PrNMI (0.6 mg/kg, i.p.) prevented the development of both acute and chronic hypersensitivity induced by GTN injections. Both CB1R and CB2R activation contributed to PrNMI’s suppression of acute GTN-induced hypersensitivity. Repeated GTN administration significantly increased ion channel, enzymes and second messenger markers of trigeminal sensitization, which were prevented by PrNMI pretreatment. Chronic GTN treatment (10 mg/kg) increased activation of low pH- and allyl isothiocyanate-induced currents in TG neurons, while PrNMI reversibly (1 M) suppressed both of these currents in TG neurons of na�ve mice. These results demonstrate the efficacy of PrNMI as a putative preventative therapeutic for chronic migraine.

Alcohol use disorder (AUD) is a chronic, relapsing disorder that represents a substantial societal burden at a global scale. In the US alone, AUD is associated with societal and financial costs exceeding $249 billion per year. While many use alcohol for its reported anxiolytic and gregarious properties, consuming alcohol directly activates the hypothalamic stress axis. Persons with AUD, that are consistently exposed to this stressful stimulus, are known to have alterations in the acute response to alcohol. Little is known about how chronic ethanol exposure alters the plastic and metaplastic responses of stress, per se.Relapse remains a main barrier in the pharmaceutical treatment of AUDs. While there are some medications currently available for AUD, none explicitly target the hypothalamic-pituitary-adrenal (HPA) axis. Further, sex is an essential variable to consider when pharmacologically targeting the HPA axis, as both acute and developmental effects of gonadal hormones can influence neuronal signaling in various brain regions. In Chapter 1, I investigate the effect of chronic intermittent vapor exposure to progressively increasing doses of ethanol (CIE) followed by protracted withdrawal on the physiological response to norepinephrine (NE; 10�M). I also investigate the mechanistic contributions of specific α adrenergic receptors (AR; α1AR & α2AR) on the CIE-induced neurophysiological effects. The paraventricular nucleus of the hypothalamus initiates a robust glutamate signal to start the overcome tonic GABAergic inhibition and adaptively coordinate stress-associated behaviors. We have previously shown that this metaplastic feature is disrupted in gavage models of CIE due to altered function of N-methyl D-aspartate receptor. In Chapter 2, I demonstrate that the CIEvapor model produces a similar impairment in hypothalamic metaplasticity. I also demonstrate how this effect is impacted by blockade of α1AR (with prazosin; 10�M). Astrocytes and neurons undergo a complex interplay in response to changes in the chemical gradient of various neurotransmitters. Both neurons and astrocytes have distinct responses during acute alcohol intoxication and protracted withdrawal. In Chapter 3, I investigate differences in how astrocytes and/or neurons may impact the noradrenergic activation and subsequent glucocorticoid receptor mediated inhibition of the HPA axis. The tripartite synapse is very likely involved in the regulation of stimulation-induced plasticity due to the critical role astrocytes play in glutamate shuttling, neurotransmitter release and regulation of synaptic activity. Despite this, we have not found studies evaluating the contribution of astrocytes to the metaplastic short-term glutamatergic potentiation onto CRF neurons following high-frequency stimulation. In Chapter 4, I investigate cell-specific differences to spontaneous Ca2+ events and event kinetics following CIEvapor. Various preclinical models of AUD can reliably induce anxiety-like behavior. However, AUD models fail to have full penetrance of ethanol (EtOH) preference and escalation of consumption behaviors. There are also noted genetic differences that contribute to EtOH preference. In Chapter 5, I investigate differences to stress-associated and consummatory alcohol behaviors following CIEvapor treatment. I also investigate the convergence and divergence of stress-related and consummatory behaviors with neurophysiological features of ex vivo Ca2+ signals

Chronic migraine is debilitating and greatly diminishes individual’s quality of life. Moreover, repeated use of current abortive medications leads to rebound headaches and latent sensitization of trigeminal ganglion (TG) neurons. Emerging evidence indicates that cannabinoids may be effective in reducing the frequency of migraine attacks. However, their widespread use is limited by their psychotropic side effects. We recently developed peripherally-restricted cannabinoids (PRCBs), demonstrating their effectiveness in several preclinical models of neuropathic and cancer pain, without psychotropic side effects. We investigated the efficacy of our lead PRCB in a mouse model of medication overuse headache and latent sensitization, assessed changes in protein expression of select ion channels and enzymes involved in the pathogenesis of these headaches, and examined the cellular mechanisms by which peripheral cannabinoid 1 receptor (CB1R) activation exerts its effects on TG neurons. Pre-treatment with a PRCB prevented chronic triptan-induced hypersensitivity, latent sensitization, and increases in phosphorylated protein kinase A, neuronal nitric oxide synthase, and transient receptor ankyrin 1 protein levels compared to vehicle-treated mice. PRCB reversibly suppressed low pH- and allyl isothiocyanate-evoked currents in TG neurons in a CB1R-dependent manner. PRCB effectiveness in preventing triptan-induced latent sensitization points to its potential as a preventative treatment for rebound headaches.