As a major public health problem affecting more that 76.5 million Americans, chronic pain is one main reason why people seek medical attention. It is a pathological nervous system disorder that persists for months or years. Sensitization of nociceptive neurons in the dorsal horn of the spinal cord is crucial in the development of allodynia and hyperalgesia. The work presented in this thesis will focus on spinal protein kinase M zeta (PKMζ)-mediated plasticity and GABAergic disinhibition as spinal amplification mechanisms that orchestrate persistent changes in the dorsal horn of the spinal cord. As a result of central sensitization following peripheral nerve injruy, GABAergic disinhibition occurs due to an alteration in Cl- homeostasis via reduced KCC2 expression and function. Intrathecal administration of acetazolamide (ACT), a carbonic anhydrase inhibitor, attenuated neuropathic allodynia and spinal co-adminitation of ACT and midazolam (MZL), an allosteric modulator of the benzodiazepine class of GABAA receptors, synergistically inhibited neuropathic allodynia. Further studies concerning the impact of altered Cl-homeostasis via reduced KCC2-mediated Cl-extrusion capacity on the analgesic efficacy and potency of GABAA receptor agonist and allosteric modulators revealed that there is a differential regulation of the agonists and allosteric modulators at the GABAA receptor complex when Cl-homeostasis is altered. Another spinal amplification mechanism leading to central sensitization is PKMζ-mediated spinal LTP. In model of persistent nociceptive sensitization, allodynia induced by IL-6 injection or plantar incision was abolished by both the inhibition of protein translation machinery and PKMζ inhibitor, ZIP. However, only PKMζ inhibition prevented the enhanced pain hypersensitivity precipitated by a subsequent stimulus after the initial hypersensitivity had resolved, asserting that spinal PKMζ underlies the maintenance mechanisms of persistent nociceptive sensitization. Also, these results confirmed that the initiation mechanisms of persistent sensitization parallel LTP initiation mechanisms and the maintenance mechanisms of persistent sensitization parallel LTP maintenance mechanisms. Taken together, these results indicate that these amplification mechanisms drive a chronic persistent state in these models such that inhibition of these spinal amplication mechanisms will serve as an effective approach in the quenching chronic pain hypersensitivity in chronic pain models.

Learning and memory has been a topic that has captured the attention of the scientific and public communities since the dawn of scientific discovery. Without the faculty of memory, mammals cannot experience nor function in the world; among homosapiens specifically, language, relationships, and personal identity cannot be developed (Eysenck, 2012). After all, some philosophers such as John Locke argued we are nothing but a collection of past memories in which we have developed and improved upon (Nimbalkar, 2011). Understanding the cellular mechanisms behind learning, and the subsequent formation of memory, has been a topic that has garnered scientific interest for many decades. One particular kinase that has been at the center of attention in the last decade is the serine/threonine kinase PKM-ζ, an N-terminal truncated form of PKC-ζ that renders it constitutively active (Hernandez et al., 2003). PKM-ζ has long been implicated in a cellular correlate of learning, long-term potentiation (LTP). Inhibition of PKM-ζ with Zeta-inhibitory peptide (ZIP) has been shown in many brain structures to disrupt maintenance of AMPA receptors, irreversibly disrupting numerous forms of learning and memory that have been maintained for weeks. The voltage-gated potassium channel Kv1.2 is a critical modulator of neuronal physiology, including dendritic excitability, action potential propagation, and neurotransmitter release. While expressed in various mammalian tissues, Kv1.2 is most prevalent in the cerebellum where it modulates both dendritic excitability of Purkinje cells (PCs) and basket cell (BC) inhibitory input to PCs. Because PCs are the main computational unit of the cerebellar cortex and provide its sole output (Napper et al., 1988; Harvey et al., 1991), regulation of synaptic Kv1.2 is predicted to have a major role in cerebellar function. Pharmacological inhibition of Kv1.2 in cerebellar PC dendrites increases excitability (Khavandgar et al., 2005), while its inhibition in BC axon terminals increases inhibition to PCs (Southan & Robertson, 1998). Interestingly, two prior studies have demonstrated that PKC-ζ, an atypical Protein Kinase C, is able to phosphorylate and bind cerebellar Kvβ2, a Kv1.2 auxiliary subunit. (Gong et al., 1999; Croci et al., 2003). Delay eyeblink conditioning (EBC) is an established model for the assessment of cerebellar learning. Despite being highly expressed in the cerebellum, no studies have examined how regulation of cerebellar PKM-ζ may affect cerebellar-dependent learning and memory nor have they examined the possible effect PKM-ζ may have on Kv1.2. The goal of this dissertation was to determine whether PKM-ζ could modulate EBC in a Kv1.2 dependent manner. Through the use of microscopy techniques we have shown that PKM-ζ is highly expressed in the cerebellar cortex, primarily in the PC, and by the use of pharmacological manipulations, it was found that PKM-ζ has an important role in regulating the acquisition of EBC. Through the use of biotinylation, flow cytometry, and behavioral manipulations, it was determined that PKM-ζ regulates Kv1.2 during eyeblink conditioning. These studies provided the first evidence that PKM-ζ has a role for learning and memory in the cerebellum, and the first evidence of PKM-ζ regulating a voltage-gated ion channel rather than a ligand-gated ion channel such as AMPA receptors.