Michelle Vincler

Molecular mechanism for analgesia involving specific antagonism of α9α10 nicotinic acetylcholine receptors

α9α10 nicotinic acetylcholine receptors (nAChRs) have been identified in a variety of tissues including lymphocytes and dorsal root ganglia; except in the case of the auditory system, the function of α9α10 nAChRs is not known. Here we show that selective block (rather than stimulation) of α9α10 nAChRs is analgesic in an animal model of nerve injury pain. In addition, blockade of this nAChR subtype reduces the number of choline acetyltransferase-positive cells, macrophages, and lymphocytes at the site of injury. Chronic neuropathic pain is estimated to affect up to 8% of the worlds population; the numerous analgesic compounds currently available are largely ineffective and act through a small number of pharmacological mechanisms. Our findings not only suggest a molecular mechanism for the treatment of neuropathic pain but also demonstrate the involvement of α9α10 nAChRs in the pathophysiology of peripheral nerve injury.

Patterns of cocaine self-administration in rats produced by various access conditions under a discrete trials procedure

Frequency of drug access greatly affects the pattern and stability of cocaine self-administration. Previous research has shown that restricted drug availability produces remarkably consistent levels of daily cocaine intake, whereas increased or unlimited access produces more variable patterns of self-administration that may change over time. In the present study we used a discrete trials (DT) procedure to document how levels of access affect the pattern of cocaine intake. Rats that had been implanted with a chronically indwelling jugular cannula and trained to self-administer cocaine on an FR1 schedule were given access to cocaine during 10-min DT that were initiated throughout the day/night cycle for 21 days. Frequency of access (2, 3, 4 or 5 trials/h; 1.5 mg/kg/inj) and dose (1.0, 1.5, 2.0 and 2.5 mg/kg/inj, 3 trials/h) were investigated in separate groups of rats. When rats were presented with two or three trials an hour (1.5 mg/kg/inj), a highly regular and circadian pattern of intake was observed across weeks. Increasing the number of trials or increasing the unit dose resulted in a significant increase in average daily cocaine intake. Access to higher dose or higher frequency conditions produced a sustained drug-taking binge during the first few days on the schedule. Rats given access to five trials per hour typically responded at every opportunity for more than 48 h, then stabilized within a range of 80-100 mg/kg/day for the remainder of the experiment. To assess whether such high levels of cocaine intake had altered the motivation to respond, cocaine reinforced break-points were assessed on a progressive ratio schedule (0.32, 1.5 and 3.0 mg/kg/inj) in separate groups of animals before and 24 h after 5 days access on the DT procedure (5 trial/h). Sustained exposure to high levels of cocaine produced a shift in the dose-response curve to the right indicating tolerance to the reinforcing effects. This DT procedure provides a method to examine the behavioral and neurochemical effects of high cocaine intake over extended periods without toxicity.

Alpha9 nicotinic acetylcholine receptors and the treatment of pain.

Chronic pain is a vexing worldwide problem that causes substantial disability and consumes significant medical resources. Although there are numerous analgesic medications, these work through a small set of molecular mechanisms. Even when these medications are used in combination, substantial amounts of pain often remain. It is therefore highly desirable to develop treatments that work through distinct mechanisms of action. While agonists of nicotinic acetylcholine receptors (nAChRs) have been intensively studied, new data suggest a role for selective antagonists of nAChRs. alpha-Conotoxins are small peptides used offensively by carnivorous marine snails known as Conus. A subset of these peptides known as alpha-conotoxins RgIA and Vc1.1 produces both acute and long lasting analgesia. In addition, these peptides appear to accelerate the recovery of function after nerve injury, possibly through immune mediated mechanisms. Pharmacological analysis indicates that RgIA and Vc1.1 are selective antagonists of alpha9alpha10 nAChRs. A recent study also reported that these alpha9alpha10 antagonists are also potent GABA-B agonists. In the current study, we were unable to detect RgIA or Vc1.1 binding to or action on cloned GABA-B receptors expressed in HEK cells or Xenopus oocytes. We review the background, findings and implications of use of compounds that act on alpha9* nAChRs.(1).

Neuronal nicotinic receptors as targets for novel analgesics.

The potential use of nicotinic acetylcholine receptor agonists has been the subject of a number of recent reviews. Despite the promises of better things to come, few new compounds have been identified that circumvent the issues hindering the widespread use of the previously described nicotinic analgesics, mainly a narrow therapeutic window between analgesic efficacy and toxicity, and a lack of knowledge of native nicotinic acetylcholine receptor expression. However, several recent developments have potentially opened new windows of opportunity in the use of nicotinic agents for analgesia. A small number of laboratories have reported that peripheral nerve injury alters the pharmacology of nicotinic receptors, resulting in a leftward shift of analgesic potency but not of toxicity. Another important development in the pathophysiology of neuropathic pain is the reliance of nerve injury-induced behavioural hypersensitivity on both peripheral and central neural immune interactions. Finally, the reported neuroprotective effects of nicotine following spinal cord injury may provide an opportunity for the development of selective nicotinic agonists that are capable of attenuating chronic pain. The current review will attempt to highlight these recent developments and outline key findings that demonstrate further opportunity for the development of nicotinic agonists as novel analgesics.

Subtype-selective conopeptides targeted to nicotinic receptors:Concerted discovery and biomedical applications

Conus peptides that are selectively targeted to different molecular isoforms of nicotinic acetylcholine receptors (nAChRs) have been identified and characterized; several have recently been shown to have significant biomedical potential. An emerging strategy for the discovery from animal biodiversity of subtype-specific ligands for ion channel families is described in this review. Characterization of the gene family encoding a set of related ligands is required for discovery using a molecular genetics approach; when discovery is guided by a knowledge of the phylogeny of the biodiverse animal lineage being used as a source of ligands, a rational, efficient scan of the library of putative ligands becomes feasible. Together, these constitute an approach to uncover subtype-specific ligands, called “concerted discovery”; this was applied to the α-conotoxins, a family of Conus peptides generally targeted to nAChRs.

Targeting the α9α10 nicotinic acetylcholine receptor to treat severe pain

The α9α10 nicotinic acetylcholine receptors (nAChRs) are recognized for their function in the hair cells of the inner ear; transcripts for a9 and/or a10 subunits have also been identified in a diverse range of other tissues , including immune cells. The functioning of α9α10 nAChRs in these latter tissues is unknown. However, a recent series of studies has provided evidence that blockade of the α9α10 nAChR can alleviate chronic pain resulting from overt peripheral nerve injury or inflammation and increase the functional recovery of damaged neurons. Systemic administration of α9α10 antagonists produces an acute analgesia; repeated daily administrations produces sustained and cumulative levels of analgesia across 7 days without the development of tolerance. Although the exact mechanism of action is unknown, antagonism of the α9α10 nAChRs reduces the number of immune cells present at the site of injury.

Estrous cycle modulation of nociceptive behaviors elicited by electrical stimulation and formalin

The impact of circulating ovarian hormones on nociceptive behaviors elicited by phasic and tonic stimuli was evaluated in rats using two behavioral tests: an operant escape task and the formalin test. The operant escape task was structured to separately evaluate hindlimb flexion reflexes, the latency of escape, and the amplitude of peak vocalization to a series of phasic electrocutaneous stimuli (0.05-0.8 mA), whereas the formalin test evaluated nociceptive behaviors elicited by tonic stimulation following a subcutaneous injection of dilute formalin (1%). Hindlimb reflex amplitude, escape latency, and peak vocalization varied across the estrous cycle, such that rats were most sensitive to electrical stimuli during proestrus (reflex and escape latency) and diestrus (vocalization). Furthermore, morphine-induced (3 mg/kg sc) attenuation of hindlimb reflex amplitude was sensitive to estrous cycling. During proestrus, morphine produced less attenuation of hindlimb reflex amplitude than during nonproestrus phases. However, estrous cycling did not alter nociceptive behaviors elicited by 1% formalin. These data support the notion that circulating ovarian hormones may differentially modulate behaviors associated with phasic and tonic pain.

The effect of peripherally administered CDP-choline in an acute inflammatory pain model: the role of alpha7 nicotinic acetylcholine receptor.

BACKGROUND: CDP-choline (citicholine; cytidine-5′-diphosphate choline) is an endogenously produced nucleotide which, when injected intracerebroventricularly, exerts an antinociceptive effect in acute pain models mediated by central cholinergic mechanisms and &agr;7 nicotinic acetylcholine receptors (&agr;7nAChR). Previous reports also suggest that the peripheral cholinergic system has an antiinflammatory role mediated by &agr;7nAChRs on macrophages. METHODS: We used male Sprague-Dawley rats to assess the antihypersensitivity and antiinflammatory effect of CDP-choline after intraplantar injection of carrageenan (100 &mgr;L, 2%). Mechanical paw withdrawal thresholds and paw thickness were measured by Randall-Selitto testing and microcallipers, respectively. All drugs were administered intraplantarly in a volume 50 &mgr;L. RESULTS: CDP-choline (1, 2.5, 5 &mgr;mol; intraplantar) increased the mechanical paw withdrawal threshold and decreased paw edema in a dose- and time-dependent manner in the carrageenan-injected hindpaw. CDP-choline administration to the noninflamed contralateral hindpaw did not alter ipsilateral inflammation. Methyllycaconitine (100 nmol), a selective &agr;7nAChR antagonist, completely blocked the effects of CDP-choline when administered to the inflamed hindpaw. However, the administration of methyllycaconitine to the contralateral hindpaw did not block the effects of CDP-choline in the ipsilateral paw. The administration of CDP-choline (5 &mgr;mol) 10 min after carrageenan administration to the ipsilateral hindpaw did not reduce swelling and edema but did significantly reduce hypersensitivity. Treatment with CDP-choline decreased tumor necrosis factor-&agr; production in the rat paw tissue after carrageenan. CONCLUSIONS: The results of this study suggest that intraplantar CDP-choline has antihypersensitivity and antiinflammatory effects mediated via &agr;7nAChRs in the carrageenan-induced inflammatory pain model.

Inhibitory M2 muscarinic receptors are upregulated in both axotomized and intact small diameter dorsal root ganglion cells after peripheral nerve injury.

Acetylcholine reduces nociceptive input in part by activating inhibitory M2 muscarinic receptors on primary sensory neurons, and acetylcholinesterase inhibitors and muscarinic agonists produce analgesia in humans and animals. M2 muscarinic receptors are upregulated in animals with diabetic neuropathy, but their level of expression and function after peripheral nerve injury has not been previously examined. This study tested, using intracellular Ca(2+) response to membrane depolarization, the effect of the M2 muscarinic receptor agonist bethanechol on individual dorsal root ganglion cells from normal and L5-6 spinal nerve-ligated rats, followed by M2 muscarinic receptor immunostaining. We also examined functional transient receptor potential for vanilloids-1 activity by determining intracellular Ca(2+) response evoked by capsaicin in M2 muscarinic receptor immunoreactive cells. In normal dorsal root ganglion cells, bethanechol inhibited the Ca(2+) response in a concentration-related fashion, and this inhibition was blocked by the M2 muscarinic receptor antagonist gallamine. Cells expressing M2 muscarinic receptors by immunostaining were significantly inhibited by bethanechol, whereas those lacking positive staining were not. The proportion of studied dorsal root ganglion neurons with positive M2 muscarinic receptor staining increased significantly in the injured ipsilateral L5-6 and the uninjured ipsilateral L4 ganglia, but not in the contralateral dorsal root ganglion neurons compared with normals. In contrast, the proportion of neurons responding to capsaicin significantly decreased in the injured ipsilateral L5-6 dorsal root ganglion cells. These results suggest that inhibitory M2 muscarinic receptors are upregulated in small- and medium-sized axotomized dorsal root ganglion neurons and their uninjured neighbors following nerve injury, and may represent an appropriate target for analgesia in this setting.

Immunocytochemical localization of the α3, α4, α5, α7, β2, β3 and β4 nicotinic acetylcholine receptor subunits in the locus coeruleus of the rat

The presence of nicotinic acetylcholine receptors (nAChRs) within the locus coeruleus (LC) has been examined using a wide range of techniques. However, the expression pattern of individual nicotinic receptor subunits has not been described. Using immunocytochemistry, we demonstrate the distribution of the alpha3, alpha4, alpha5, alpha7, beta2, beta3 and beta4 nAChR subunits within the LC. Most nAChR subunits were expressed on neuronal perikarya within the LC nucleus. The alpha3, alpha4, alpha7 and beta3 immunoreactive neurons were evenly distributed in the dorsal and ventral LC whereas the alpha5, beta2 and beta4 nAChR subunits were preferentially confined to the upper dorsal section. In addition to neuronal perikarya, alpha4, alpha5 and beta2 immunoreactive fibers were observed. With the exception of the alpha3 subunit, punctate labeling was observed within and immediately surrounding the LC. These data are consistent with the presence of multiple nAChRs within the LC and extend these findings to show the distribution pattern of each nAChR subunit throughout the LC nucleus.