• Dural Afferent Mechanisms of Migraine Pain

      Harriott, Andrea; Gold, Michael S., Ph.D. (2009)
      Compelling evidence indicates that dural afferent sensitization initiates migraine pain. Furthermore, mechanisms of dural afferent sensitization may be unique given that ion channels underlying afferent excitability differ greatly depending on the target of innervation. Triptans, serotonin 1B/1D receptor (5HT1B/1DR) agonists, are selectively used for migraine pain which raises the possibility that their selectivity reflects modulation of unique mechanisms of dural afferent sensitization. The specific hypotheses tested in my thesis were that: 1) triptan selectivity reflects differential 5HT1DR distribution, 2) dural afferents have unique electrophysiological properties, and 3) triptan selectivity reflects modulation of these unique properties. Female Sprague Dawley rats were used for all experiments. The density of 5HT1DR was quantified with Western blot and localized with immunohistochemistry. Acutely dissociated dural afferents were examined using patch clamp recordings and compared to temporalis muscle afferents (TM) in the absence and presence of inflammatory mediators (IM): (μM) prostaglandin E2 (1), bradykinin (10), and histamine (1); and sumatriptan. There were four major observations in this thesis : 1) The 5HT1DR is differentially distributed in nerve fibers innervating peripheral tissues such that the density is highest in tissues known to produce migraine-like pain (i.e. circle of Willis and dura) than in structures in which triptans have no efficacy (i.e. TM), 2) dural afferents have marked differences in the ion channel mechanisms mediating passive and active electrophysiological properties in the absence and presence of IM including an increase in Na+ current, a decrease in Ca2+-dependent K+ current, a decrease in voltage gated Ca2+ current, and an increase in Ca2+ dependent Cl- current, and 3) prolonged sumatriptan incubation with 1µM sumatriptan prevented dural afferent sensitization via K+ current modulation. Taken together, the importance of dural afferent signaling in migraine pathogenesis provide a rationale utilizing ionic mechanisms involved in dural afferent sensitization as targets of novel anti-migraine therapies.
    • Switch of GABAA signaling with persistent inflammation

      Zhu, Yi; Gold, Michael S., Ph.D. (2012)
      Following inflammation there is a switch in spinal GABAA signaling from inhibition to excitation such that GABA receptors contribute to inflammatory hyperalgesia. We hypothesized that this switch occurs in primary afferent neurons, as a result of a steady state and/or dynamic depolarizing shift in the reversal potential of GABAA currents (EGABA) which is coupled to an increase in high affinity extrasynaptic GABAA receptors. To test this hypothesis, back labeled, acutely dissociated cutaneous dorsal root ganglion (DRG) neurons from naïve and inflamed rats were studied with a variety of techniques including Ca2+ imaging and patch-clamp electrophysiology. With calcium imaging, GABAA receptor activation was shown to be inhibitory in neurons from naïve animal but was facilitatory or directly exciting in neurons from inflamed rats. Results from gramidicin perforated patch showed that a steady-state depolarizing shift in EGABA was not responsible for this shift in signaling. Rather, the shift appeared to be due to a combination of changes including an increase in GABAA current, a decrease in K+ current, and a depolarizing shift in resting membrane potential. The increase in GABAA current was associated with an increase in both high and low affinity currents which was due to a persistent increase in the relative activity of tyrosine kinase, resulting in part to a decrease in receptor internalization, rather than a change in subunit expression or protein. Dynamic regulation of EGABA was also observed in association with neural activity, but the shift in EGABA was hyperpolarizing, and likely to be due to the activation of a Cl- channel rather than a change in secondary ion transporter activity. Interestingly, inflammation was associated with a decrease in the activity dependent shift in EGABA. Our results indicate that the inflammation-induced switch in GABAA signaling is a complex process that involves the modulation of multiple channels and Cl- equilibrium potential and suggested different approaches to prevent the hyperalgesic effect of GABA.