Characterization of sensory neurons in the dorsal root ganglia of Bax-deficient mice.

During development, the rescue of spinal motoneurons as well as sensory neurons in the dorsal root ganglion (DRG) from programmed cell death (PCD) depends on the integrity of peripheral target innervation. Following deletion of the pro-apoptotic gene Bax, both motoneurons and DRG neurons are rescued from PCD. In the present paper, we asked whether different cell types in the DRG exhibit distinct responses to Bax deletion. In 1-month-old Bax-deficient (Bax-/-) mice, distinct subsets of DRG neurons that were immunopositive for TrkA, CGRP, TRPV1 or TrkC, were all increased in number and exhibited cell atrophy compared to wild type DRG neurons. In addition there was hyperinnervation of the epidermis by CGRP immunopositive processes and a correlated functional hypersensitivity of mechanical nociception in Bax-/- mice. By contrast, the functional properties of populations of rescued thermoreceptor and mechanoreceptor DRG neurons were unchanged. These data indicate that although Bax deletion rescues all of the DRG cell types examined here from PCD, the functional consequences of having excess cells differ between sensory phenotypes.

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).

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 alpha7 nicotinic acetylcholine receptors (alpha7nAChR). Previous reports also suggest that the peripheral cholinergic system has an antiinflammatory role mediated by alpha7nAChRs 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 microL, 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 microL. RESULTS: CDP-choline (1, 2.5, 5 micromol; 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 alpha7nAChR 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 micromol) 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-alpha 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 alpha7nAChRs in the carrageenan-induced inflammatory pain model.

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 alpha-conotoxins, a family of Conus peptides generally targeted to nAChRs. Subtype-specific alpha-conotoxins were developed that target two groups of nAChRs, alpha(6)* and alpha(9)*. alpha-conotoxin MII has become the defining ligand for identifying the alpha(6)* nAChR subtype. A synthetic analog, MII [E11A], further subdivides alpha(6)* nAChRs into those that contain an alpha(4) subunit and those that do not. Importantly, these two subtypes are differentially affected by nigrostriatal damage, findings of likely relevance to the pathopysiology of Parkinson's disease. In contrast, alpha-conotoxins that target alpha(9) nAChR subtypes have potential as analgesics for the treatment of neuropathic pain that develops after nerve injury. The discovery of alpha-conotoxin RgIA enabled the identification of a novel role for alpha(9)* nAChRs. Use of alpha(9)* nAChR antagonists is associated with reversal of inflammation caused by the nerve injury. Thus, subtype-specific alpha-conotoxins targeted to particular nAChR isoforms are not only useful for understanding the physiological role of these receptors, but can have important diagnostic and therapeutic applications as well.

Spinal alpha3beta2* nicotinic acetylcholine receptors tonically inhibit the transmission of nociceptive mechanical stimuli.

The presence of non-alpha4beta2, non-alpha7 nicotinic acetylcholine receptors (nAChR) in the rat spinal cord has been suggested previously, but the identity of these nAChRs had not been shown. Intrathecal administration of the alpha3beta2*/alpha6beta2* selective alpha-conotoxin MII (alpha-CTX MII) dose- and time-dependently reduced paw withdrawal thresholds to mechanical pressure in normal rats. The pronociceptive effect of alpha-CTX MII was partially blocked by NMDA receptor antagonism and lost completely following ablation of C-fibers. The effect of spinal nerve ligation on alpha-CTX MII-induced mechanical hypersensitivity was also assessed. Sensitivity was lost in the hind paw ipsilateral to spinal nerve ligation, but maintained in the contralateral hind paw at control levels. Radioligand binding in spinal cord membranes revealed high and low affinity alpha-CTX MII binding sites. Spinal nerve ligation did not significantly alter alpha-CTX MII binding ipsilateral to ligation. Finally, no evidence for the presence of alpha6-containing nAChRs was identified. The results of these studies show the presence of 2 populations of alpha-CTX MII-sensitive nAChRs containing the alpha3 and beta2, but not the alpha6, subunits in the rat spinal cord that function to inhibit the transmission of nociceptive mechanical stimuli via inhibiting the release of glutamate from C-fibers. Spinal nerve ligation produces a unilateral loss of alpha-CTX MII-induced mechanical hypersensitivity without altering alpha-CTX MII binding sites. Our data support a peripheral injury-induced loss of a cholinergic inhibitory tone at spinal alpha3beta2* nAChRs, without the loss of the receptors themselves, which may contribute to mechanical hypersensitivity following spinal nerve ligation.

Peripheral nerve injury alters spinal nicotinic acetylcholine receptor pharmacology.

Nicotinic acetylcholine receptors are widely expressed in the rat spinal cord and modulate innocuous and nociceptive transmission. The present studies were designed to investigate the plasticity of spinal nicotinic acetylcholine receptors modulating mechanosensitive information following spinal nerve ligation. A tonic inhibitory cholinergic tone mediated by dihydro-beta-erythroidine- (DHbetaE) and methyllycaconitine- (MLA) sensitive nicotinic acetylcholine receptors was identified in the normal rat spinal cord and cholinergic tone at both populations of nicotinic acetylcholine receptors was lost ipsilateral to spinal nerve ligation. The administration of intrathecal nicotinic acetylcholine receptor agonists reduced mechanical paw pressure thresholds with a potency of epibatidine=A-85380>>nicotine>choline in the normal rat. Following spinal nerve ligation, intrathecal epibatidine and nicotine produced an ipsilateral antinociception, but intrathecal A-85380 and choline did not. The antinociceptive response to intrathecal nicotine was blocked with the alpha7 and alpha9alpha10-selective nicotinic acetylcholine receptor antagonist, MLA, and the alphabeta heteromeric nicotinic acetylcholine receptor antagonist, DHbetaE. The antinociceptive effects of both intrathecal nicotine and epibatidine were mediated by GABA(A) receptors. Spinal [(3)H]epibatidine saturation binding was unchanged in spinal nerve-ligated rats, but spinal nerve ligation did increase the ability of nicotine to displace [(3)H]epibatidine from spinal cord membranes. Spinal nerve ligation altered the expression of nicotinic acetylcholine receptor subunits ipsilaterally, with a large increase in the modulatory alpha5 subunit. Taken together these results suggest that pro- and antinociceptive populations of spinal nicotinic acetylcholine receptors modulate the transmission of mechanosensitive information and that spinal nerve ligation-induced changes in spinal nicotinic acetylcholine receptors likely result from a change in subunit composition rather than overt loss of nicotinic acetylcholine receptor subtypes.

Targeting the alpha9alpha10 nicotinic acetylcholine receptor to treat severe pain.

The alpha9alpha10 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 alpha9alpha10 nAChRs in these latter tissues is unknown. However, a recent series of studies has provided evidence that blockade of the alpha9alpha10 nAChR can alleviate chronic pain resulting from overt peripheral nerve injury or inflammation and increase the functional recovery of damaged neurons. Systemic administration of alpha9alpha10 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 alpha9alpha10 nAChRs reduces the number of immune cells present at the site of injury.

Increased spinal dynorphin contributes to chronic nicotine-induced mechanical hypersensitivity in the rat.

Chronic nicotine administration has been shown previously to produce mechanical hypersensitivity in the rat although the mechanism of this effect is unknown. Rats treated with chronic systemic nicotine 3.6 or 8.6 mg/(kg day) for 14-21 days displayed mechanical hypersensitivity coincident with an increase of prodynorphin immunoreactivity and dynorphin content within the spinal cord. The administration of dynorphin antiserum intrathecally significantly attenuated chronic nicotine-induced mechanical hypersensitivity. Our results suggest that chronic nicotine administration produces an increase in spinal dynorphin content and release that contributes to mechanical hypersensitivity.

Impact of chronic nicotine on sciatic nerve injury in the rat.

Chronic nicotine exposure and the immune response to peripheral nerve injury has not been investigated thoroughly. Rats were exposed to chronic nicotine or saline followed by chronic constriction injury (CCI) of the sciatic nerve. Mechanical sensitivity was measured at various time points and the immune response was investigated at 21 days post-CCI. Chronic nicotine increased mechanical hypersensitivity, microglia activation, and the production of IL-1beta, but not the number of immune cells at the site of injury. These results suggest that chronic nicotine increases mechanical hypersensitivity following peripheral nerve injury through a mechanism that may involve an increased production and release of central and peripheral cytokines.

Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10 nicotinic acetylcholine receptors.

alpha9alpha10 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 alpha9alpha10 nAChRs is not known. Here we show that selective block (rather than stimulation) of alpha9alpha10 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 world's 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 alpha9alpha10 nAChRs in the pathophysiology of peripheral nerve injury.

Spinal glial activation contributes to postoperative mechanical hypersensitivity in the rat.

UNLABELLED: The activation of spinal cord microglia and astrocytes after peripheral nerve injury or inflammation contributes to behavioral hypersensitivity. The contribution of spinal cord glia to mechanical hypersensitivity after hind paw incision has not been investigated previously. Male Sprague-Dawley rats underwent a unilateral plantar hind paw incision, and the development of mechanical hypersensitivity was assessed by using von Frey filaments. The activation of spinal cord microglia and astrocytes was measured 1, 2, 3, and 5 days after hind paw incision by using immunohistochemistry. The glial activation inhibitor, fluorocitrate, was administered intrathecally 24 hours after hind paw incision to determine glial involvement in mechanical hypersensitivity. Hind paw incision induced an activation of spinal astrocytes ipsilateral to incision within 24 hours. Both microglia and astrocytes reached a maximum activation 3 days after hind paw incision. Fluorocitrate produced a dose-dependent reduction in mechanical hypersensitivity when administered 24 hours after hind paw incision. Spinal cord glial activation contributes to the mechanical hypersensitivity that develops after hind paw incision. PERSPECTIVE: Hind paw incision produces mechanical hypersensitivity that can be alleviated with the inhibition of spinal cord glia. Our results suggest that the activation of spinal cord astrocytes within 24 hours of incision contributes to mechanical hypersensitivity. Therefore, spinal cord astrocytes might represent a novel target for the treatment of postoperative pain.

Impact of chronic nicotine on the development and maintenance of neuropathic hypersensitivity in the rat.

RATIONALE: Clinical data support a correlation between smoking and the incidence and severity of some chronic pain conditions. However, the impact of nicotine on neuropathic pain has been largely ignored in the laboratory setting. OBJECTIVES: The purpose of these studies was to determine if chronic nicotine would alter mechanical hypersensitivity after spinal nerve ligation. MATERIALS AND METHODS: Rats were implanted with osmotic mini pumps to administer either saline or nicotine (4, 10, or 24 mg/kg/day) for 7 or 21 days. On day 7 of saline/nicotine administration, rats receiving 24 mg/kg/day nicotine underwent spinal nerve ligation. Mechanical thresholds to pressure were measured across nicotine exposure and spinal cords were collected on days 7 or 21. Spinal cord slices were immunostained for phosphorylation of cAMP response element binding protein (pCREB), to determine general neuronal activity, and for cleaved caspase-3, as a marker for apoptosis. RESULTS: Chronic nicotine produced a dose-dependent and stable mechanical hypersensitivity, which could be blocked with the alpha4beta2-selective antagonist, dihydro-beta-erythroidine (DHbetaE). Spinal nerve ligation also produced a stable mechanical hypersensitivity, which was exacerbated in the presence of chronic nicotine. Differences in mechanical sensitivity were reflected in spinal pCREB, which was highly correlated with the degree of mechanical hypersensitivity. Chronic nicotine also altered the number of pro-apoptotic cells in the spinal cord as measured by cleaved caspase-3. CONCLUSIONS: These findings demonstrate that chronic nicotine produces a stable, long-lasting, mechanical hypersensitivity that exacerbates mechanical sensitivity resulting from peripheral nerve injury. The mechanism of this may involve an increase in spinal neuronal activity and apoptosis.

Spinal cord dynorphin expression increases, but does not drive microglial prostaglandin production or mechanical hypersensitivity after incisional surgery in rats.

Spinally released dynorphin contributes to hypersensitivity from nerve injury, inflammation, and sustained morphine treatment, but its role in post-operative pain has not been tested. Intrathecal injection of dynorphin activates cyclooxygenase (COX)-1 and -2 to induce hypersensitivity. Spinal COX-1 expression and activity increase following incisional paw surgery in rats, although the stimulus for this increase is not known. In the current study we tested whether spinal dynorphin expression increases after incisional surgery and induces hypersensitivity in this setting, and whether dynorphin stimulates COX-1 activity in spinal cord microglia. Paw incision resulted in increased prodynorphin immunoreactivity in laminae I, IIo, and V in the L4-L6 spinal cord dorsal horn ipsilateral to surgery. Change in prodynorphin expression did not parallel that of mechanical hypersensitivity. Repeated intrathecal dynorphin A antiserum injection failed to alter mechanical hypersensitivity after incisional surgery, although it was effective against mechanical hypersensitivity following spinal nerve ligation. Paw incision increased COX-1 immunoreactivity in the L4-L6 ipsilateral spinal cord, and these cells were confirmed to be microglia by co-localization with OX-42. Spinal cord microglia in culture expressed COX-1 immunoreactivity and released PGE2, but dynorphin A failed to increase release of PGE2 in these cultures. These results suggest that increased COX-1 expression occurs in spinal cord microglia following incisional surgery. Although prodynorphin immunoreactivity also increases, it likely does not drive COX-1 expression or mechanical hypersensitivity in this setting.

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.

Knock down of the alpha 5 nicotinic acetylcholine receptor in spinal nerve-ligated rats alleviates mechanical allodynia.

Nicotinic acetylcholine receptor (nAChR) agonists are known to alleviate neuropathic and inflammatory pain via activation of a heterogeneous population of receptors. However, the function of nAChRs in the maintenance of neuropathic pain is not known. Spinal nerve ligation (SNL) increases the spinal expression of the alpha5 nAChR subunit ipsilateral to injury. The alpha5 subunit is unique because it modifies numerous characteristics of existing functional nAChRs, but it does not form functional nAChRs when expressed alone or with beta nicotinic subunits. Because there are no alpha5 subunit selective ligands, we used antisense oligonucleotides (ODNs) to assess the contribution of the alpha5 subunit to the maintenance of mechanical allodynia following SNL. Intrathecal antisense oligonucleotides were administered to SNL rats after the development of mechanical allodynia (10-12 days post-SNL). I.t. antisense specifically reduced alpha5 immunoreactivity (alpha5-IR) by 50-70% in the outer laminae of the dorsal horn and moderately alleviated mechanical allodynia. Furthermore, using the phosphorylation of cAMP response element-binding protein (pCREB) as a general marker of neuronal activation, a significant increase in pCREB immunoreactivity was observed in SNL rats. Treatment of SNL rats with alpha5-antisense significantly reduced pCREB immunoreactivity. These results suggest that the increased expression of the alpha5 nAChR subunit following SNL contributes to spinal CREB phosphorylation and the maintenance of mechanical allodynia.

Plasticity of spinal nicotinic acetylcholine receptors following spinal nerve ligation.

The nicotinic cholinergic system is known to be important in the processing of nociceptive information. In the spinal cord, nicotinic receptors are expressed on primary afferent terminals, inhibitory interneurons and descending noradrenergic and serotoninergic fibers. Following peripheral nerve injury, the expression of numerous receptors involved in nociceptive processing is altered in the superficial dorsal horn of the spinal cord. However, the expression of nicotinic acetylcholine receptor subunits in the lumbar spinal cord following peripheral nerve injury has not been investigated. We examined the expression of the alpha3, alpha4, alpha5, alpha7, beta2, beta3 and beta4 nicotinic subunits in the spinal cord of normal and spinal nerve ligated rats using immunocytochemistry. Two nicotinic subunits were found to have an increased expression following spinal nerve ligation. The number of cells expressing the alpha3 subunit in the dorsal horn increased bilaterally following spinal nerve injury. Also, the number of alpha5 immunoreactive fibers increased significantly ipsilateral to ligation. The expression of the alpha4, alpha7, beta2, beta3 and beta4 subunits was unchanged. We propose that the increased expression of the alpha3 and alpha5 nicotinic subunits may contribute to the mechanical hypersensitivity observed following spinal nerve ligation.

Immunocytochemical localization of the alpha3, alpha4, alpha5, alpha7, beta2, beta3 and beta4 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.

Clonidine-induced neuronal activation in the spinal cord is altered after peripheral nerve injury.

BACKGROUND: Alpha 2 adrenoceptor agonists produce antinociception in normal animals and alleviate mechanical allodynia in animals with nerve injury, although their mechanism of action may differ in these situations. The purpose of this study was to examine the location and number of cells in the spinal cord activated by intrathecal clonidine in these two circumstances and to test whether one class of interneurons, cholinergic, express alpha 2 adrenoceptors. METHODS: Intrathecal saline or clonidine, 10 and 30 microg, was injected in normal rats or those with mechanical allodynia following partial sciatic nerve section. Two hours later, animals were anesthetized and pericardially perfused. The number of cells in superficial and deep dorsal horn laminae at the L4-L5 level immunostained for phosphorylated cAMP response element binding protein (pCREB) were quantified. In separate studies, the authors colocalized alpha2C adrenoceptors with cholinergic neurons. RESULTS: Intrathecal clonidine increased pCREB immunoreactive cells in both superficial and deep laminae by 50-100% in normal animals. The number of pCREB immunoreactive cells increased in nerve-injured compared to normal rats. Intrathecal clonidine decreased pCREB immunoreactive cells in the deep dorsal horn of injured animals. Alpha2C adrenoceptors colocalized with cholinergic neurons in both superficial and deep dorsal horn. DISCUSSION: Previous studies suggest a shift in alpha 2 adrenoceptor subtype and the involvement of cholinergic interneurons in antinociception in the spinal cord after nerve injury. The current results suggest that intrathecal clonidine, by direct or indirect methods, increases neuronal activation in normal animals, presumably leading to net inhibition of pain signaling, whereas it reduces the increase in neuronal activity induced by nerve injury.

Peripheral nerve injury alters the alpha2 adrenoceptor subtype activated by clonidine for analgesia.

BACKGROUND: Previous studies suggest that the alpha adrenoceptor subtype is the target for spinally administered alpha -adrenergic agonists, clonidine, for pain relief. However, ST 91, a preferential alpha adrenoceptor subtype agonist, induces antinociception, and intrathecally administered alpha antisense oligodeoxynucleotide decreases antinociception induced by clonidine in the rat, suggesting non-A sites may be important as well. Therefore, the authors examined the subtype of alpha adrenoceptor activated by clonidine and ST 91 in normal rats and those with nerve injury-induced hypersensitivity. METHODS: The same mechanical stimulus was applied to normal rats and those following spinal nerve ligation, and the effect of intrathecal clonidine and ST 91 on withdrawal threshold to the stimulus was determined. To further examine subtypes, animals were spinally pretreated with vehicle, BRL 44408 (an alpha subtype-preferring antagonist), and ARC 239 (an alpha subtype-preferring antagonist). RESULTS: In normal animals, clonidine's effect was diminished by pretreatment with either antagonist, whereas ST 91's antinociceptive effect was solely blocked by pretreatment with ARC 239. In nerve-injured animals, the antihypersensitivity action of both clonidine and ST 91 was blocked by administration of ARC 239, whereas BRL 44408 was ineffective. CONCLUSIONS: These data agree with previous studies supporting that the alpha adrenoceptor is important to the antinociceptive effect of clonidine in normal animals. Nerve injury alters this and results in a total reliance on alpha adrenoceptors.