Date of Award

5-1-2013

Degree Name

Doctor of Philosophy

Department

Pharmacology

First Advisor

Faingold, Carl

Abstract

Considerable evidence suggests the involvement of the ventrolateral periaqueductal gray (PAG) in both inhibitory and facilitatory actions within modulatory pathways for pain, The PAG is a critical nucleus in these pain networks and receives and sends extensive projections within these networks. Chronic pain is a major side effect in 30-70% of cancer patients receiving chemotherapeutic regimen. Endocannabinoids, such as anandamide (AEA), are lipid neuromodulators in the CNS that affect nociception. AEA is known to activate cannabinoid (CB1) as well as transient receptor potential vanilloid 1 (TRPV1) receptors, which are involved in nociception. Previous studies have shown that PAG is a crucial site for EC- mediated analgesia. Hypothesis one of my project is blockade of AEA uptake in the PAG will induce analgesic effects, which are mediated by CB1 and/or TRPV1 receptors. My results indicate that focal bilateral microinjection of AM404, an AEA uptake inhibitor, into the PAG induced analgesia to noxious thermal stimuli (radiant heat) both in normal and paclitaxel-treated (2 mg/kg i.p. for 4 alternate days). This analgesia was mainly mediated by both CB1 and TRPV1 receptors in the PAG in normal and by CB1 receptors in paxlitaxel-treated rats. Several studies have demonstrated that post-ictal analgesia is observed following drug-induced generalized seizures in rats. My second hypothesis is that post-ictal analgesia will occur after audiogenic seizure (AGS) induction, in genetically epilepsy prone rats (GEPR-9s), and blockade of CB1 receptors in the PAG will inhibit the analgesic effects that occur during the post-ictal period. My results indicate that induction of AGS resulted in post-ictal analgesia in GEPR-9s to noxious thermal stimuli, which was blocked upon microinjection of a CB1 receptor antagonist into the PAG suggesting a crucial role of PAG in post-ictal analgesia in GEPR-9s. Hypothesis three of my project is that, neuroplastic changes will occur in the PAG, leading to increases in neuronal firing during neuropathic pain, which will be altered by analgesic drugs. To evaluate this hypothesis, I studied the firing pattern of PAG neurons in awake behaving rats to noxious thermal stimuli. This involved evaluation of extracellular single unit activity using 8 channel microwire electrodes chronically-implanted in the PAG of unrestrained awake rats and examining responses to noxious thermal stimulation (Peltier device). The results indicate the presence of the three types of neuronal populations in the PAG that exhibit either "excitatory" or "Inhibitory" responses or were "non-responsive", to noxious thermal stimulus administered to the paw. Subsequently, I also investigated effects of pentobarbital on spontaneous and thermal stimulus evoked PAG neuronal firing, as has often been done in previous pain studies. Administration of low doses of pentobarbital significantly decreased PAG spontaneous firing and evoked excitatory and inhibitory PAG neuronal responses. These results suggest that the use of barbiturates to study PAG neuronal responses might have resulted in significant un-intended modifications of the fundamental properties of PAG neuron, as compared to the unanesthetized state. The PAG is important part of pain modulatory network, but the electrophysiological characteristics of PAG neurons in chronic neuropathic pain conditions are still unclear. This issue was addressed by administering a standard chronic pain protocol, using the cancer chemotherapeutic drug, paclitaxel, (2 mg/kg i.p. for 4 alternate days) which induced chronic neuropathic pain, Ten days after treatment, mean spontaneous firing rates of PAG neurons were increased significantly after paclitaxel treatment compared to the pre-paclitaxel treatment levels. PAG neurons in the neuropathic state exhibited significantly increased excitatory neuronal responses to non-noxious stimulus, similar to noxious thermal stimulation and significantly increased excitatory-like neuronal responses as compared to pre-treatment levels. Gabapentin is an anticonvulsant that acts on N type voltage sensitive calcium channels and possesses analgesic properties in chronic pain syndromes. Gabapentin did not significantly affect PAG neuronal responses to acute pain in my initial studies However, gabapentin did produce significant changes in spontaneous and thermal stimuli evoked PAG neuronal firing in paclitaxel-treated rats. Gabapentin induced significant dose-dependent decreases in the elevated spontaneous and evoked PAG neuronal firing to both non-noxious and noxious thermal stimuli in the paclitaxel model of neuropathic pain. I also investigated effects of AM404 on spontaneous and thermal stimuli evoked PAG neuronal firing in paclitaxel-treated rats, I observed that AM404 dose-dependently inhibited elevated spontaneous and evoked PAG neuronal firing in paclitaxel-treated rats. These effects were blocked by pre-treatment with CB1 receptor antagonist (AM251) suggesting a crucial role of CB1 receptor in AM404 mediated analgesic effects. These findings suggest that paclitaxel treatment could lead to plasticity in the PAG that might contribute to generation and maintenance of neuropathic pain. These findings provide the evidence that targeting endogenous cannabinoid system or inhibiting presynaptic calcium channels may be effective in treating neuropathic pain, in part, by actions on the PAG.

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