Jessica Maddern

α-Conotoxin Vc1.1 inhibits human dorsal root ganglion neuroexcitability and mouse colonic nociception via GABAB receptors

Objective α-Conotoxin Vc1.1 is a small disulfide-bonded peptide from the venom of the marine cone snail Conus victoriae. Vc1.1 has antinociceptive actions in animal models of neuropathic pain, but its applicability to inhibiting human dorsal root ganglion (DRG) neuroexcitability and reducing chronic visceral pain (CVP) is unknown. Design We determined the inhibitory actions of Vc1.1 on human DRG neurons and on mouse colonic sensory afferents in healthy and chronic visceral hypersensitivity (CVH) states. In mice, visceral nociception was assessed by neuronal activation within the spinal cord in response to noxious colorectal distension (CRD). Quantitative-reverse-transcription-PCR, single-cell-reverse-transcription-PCR and immunohistochemistry determined γ-aminobutyric acid receptor B (GABABR) and voltage-gated calcium channel (CaV2.2, CaV2.3) expression in human and mouse DRG neurons. Results Vc1.1 reduced the excitability of human DRG neurons, whereas a synthetic Vc1.1 analogue that is inactive at GABABR did not. Human DRG neurons expressed GABABR and its downstream effector channels CaV2.2 and CaV2.3. Mouse colonic DRG neurons exhibited high GABABR, CaV2.2 and CaV2.3 expression, with upregulation of the CaV2.2 exon-37a variant during CVH. Vc1.1 inhibited mouse colonic afferents ex vivo and nociceptive signalling of noxious CRD into the spinal cord in vivo, with greatest efficacy observed during CVH. A selective GABABR antagonist prevented Vc1.1-induced inhibition, whereas blocking both CaV2.2 and CaV2.3 caused inhibition comparable with Vc1.1 alone. Conclusions Vc1.1-mediated activation of GABABR is a novel mechanism for reducing the excitability of human DRG neurons. Vc1.1-induced activation of GABABR on the peripheral endings of colonic afferents reduces nociceptive signalling. The enhanced antinociceptive actions of Vc1.1 during CVH suggest it is a novel candidate for the treatment for CVP.

Multiple sodium channel isoforms mediate the pathological effects of Pacific ciguatoxin-1

Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (NaV), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on NaV1.1–1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at NaV1.8, while the slope factor of the conductance-voltage curves was significantly increased for NaV1.7 and peak current was significantly increased for NaV1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of NaV1.8 and the tetrodotoxin-sensitive isoforms NaV1.7 and NaV1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of NaV isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation.

Cyclic analogues of α‐conotoxin Vc1.1 inhibit colonic nociceptors and provide analgesia in a mouse model of chronic abdominal pain

Patients with irritable bowel syndrome suffer from chronic visceral pain (CVP) and limited analgesic therapeutic options are currently available. We have shown that α‐conotoxin Vc1.1 induced activation of GABAB receptors on the peripheral endings of colonic afferents and reduced nociceptive signalling from the viscera. However, the analgesic efficacy of more stable, cyclized versions of Vc1.1 on CVP remains to be determined.

Chronic linaclotide treatment reduces colitis-induced neuroplasticity and reverses persistent bladder dysfunction

Irritable bowel syndrome (IBS) patients suffer from chronic abdominal pain and extraintestinal comorbidities, including overactive bladder (OAB) and interstitial cystitis/painful bladder syndrome (IC-PBS). Mechanistic understanding of the cause and time course of these comorbid symptoms is lacking, as are clinical treatments. Here, we report that colitis triggers hypersensitivity of colonic afferents, neuroplasticity of spinal cord circuits, and chronic abdominal pain, which persists after inflammation. Subsequently, and in the absence of bladder pathology, colonic hypersensitivity induces persistent hypersensitivity of bladder afferent pathways, resulting in bladder-voiding dysfunction, indicative of OAB/IC-PBS. Daily administration of linaclotide, a guanylate cyclase-C (GC-C) agonist that is restricted to and acts within the gastrointestinal tract, reverses colonic afferent hypersensitivity, reverses neuroplasticity-induced alterations in spinal circuitry, and alleviates chronic abdominal pain in mice. Intriguingly, daily linaclotide administration also reverses persistent bladder afferent hypersensitivity to mechanical and chemical stimuli and restores normal bladder voiding. Linaclotide itself does not inhibit bladder afferents, rather normalization of bladder function by daily linaclotide treatment occurs via indirect inhibition of bladder afferents via reduced nociceptive signaling from the colon. These data support the concepts that cross-organ sensitization underlies the development and maintenance of visceral comorbidities, while pharmaceutical treatments that inhibit colonic afferents may also improve urological symptoms through common sensory pathways.

MP42-05 CHRONIC ORAL ADMINISTRATION OF THE GUANYLATE CYCLASE-C AGONIST LINACLOTIDE ATTENUATES COLITIS INDUCED BLADDER AFFERENT AND DORSAL ROOT GANGLION HYPERACTIVITY

INTRODUCTION AND OBJECTIVES: Nerve growth factor (NGF) has been implicated as an important mediator to induce C-fiber bladder afferent hyperexcitability, which contributes to the emergence of neurogenic detrusor overactivity (NDO) following spinal cord injury (SCI). In this study, we examined whether NGF neutralization using anti-NGF antibody normalizes the SCI-induced changes in electrophysiological properties of capsaicin-sensitive C-fiber bladder afferent neurons in the mouse model. METHODS: In female C57BL/6 mice, the spinal cord was transected at the Th8/9 level. Two weeks later, an osmotic pump was placed subcutaneously to administer anti-NGF antibody at 10 mg/kg/h for 2 weeks. Bladder afferent neurons were labeled with axonal transport of Fast Blue (FB), a fluorescent retrograde tracer, injected into the bladder wall 3 weeks after SCI. Four weeks after SCI, freshly dissociated L6-S1 dorsal root ganglion neurons were prepared. Whole cell patch clamp recordings were then performed in FB-labeled bladder afferent neurons, and the data were compared between SCI and spinal intact (SI) mice. After recording action potentials (AP) or voltage-gated K (Kv) currents, the sensitivity of each neuron to capsaicin was evaluated. RESULTS: In capsaicin-sensitive bladder afferent neurons, the resting membrane potentials and the peak and duration of AP did not changed by SCI. On the other hand, the threshold for eliciting AP was significantly reduced in SCI vs. SI mice. Also, SCI increased the number of AP during 800 ms membrane depolarization. These SCI induced changes were reversed by NGF neutralization. SCI induced significant increases in the diameter and cell input capacitance of capsaicin-sensitive bladder afferent neurons, which were not reversed by NGF neutralization. Densities of slow decaying KA and sustained KDR currents evoked by depolarization to 0 mV were significantly reduced by SCI. NGF neutralization reversed the SCI-induced reduction in the KA current density. CONCLUSIONS: In SCI mice, NGF plays an important role in hyperexcitability of capsaicin sensitive C-fiber bladder afferent neurons due to KA current reduction. Thus, NGF-targeting therapies could be effective for treatment of afferent hyperexcitability and NDO in SCI.