Mouse strains that lack spinal dynorphin upregulation after peripheral nerve injury do not develop neuropathic pain.

Several experimental models of peripheral neuropathy show that a significant upregulation of spinal dynorphin A and its precursor peptide, prodynorphin, is a common consequence of nerve injury. A genetically modified mouse strain lacking prodynorphin does not exhibit sustained neuropathic pain after nerve injury, supporting a pronociceptive role of elevated levels of spinal dynorphin. A null mutation of the gamma isoform of protein kinase C (PKCgamma KO [knockout]), as well as an inbred mouse strain, 129S6, also does not manifest behavioral signs of neuropathic pain following peripheral nerve injury. The objective of this study was to extend our observations to these genetic models to test the hypothesis that elevated levels of spinal dynorphin are essential for the maintenance of abnormal pain. In PKCgamma wild-type mice and the outbred mouse strain ICR, ligation of the L5 and L6 spinal nerves (SNL) elicited both tactile hypersensitivity and thermal hyperalgesia. Both strains showed a significant elevation in dynorphin in the lumbar spinal dorsal horn following SNL. Spinal administration of an anti-dynorphin A antiserum blocked the thermal and tactile hypersensitivity in both strains of mice. However, the PKCgamma KO mice and the 129S6 mice (which express PKCgamma) did not show abnormal pain after SNL; neither strain showed elevated levels of spinal dynorphin. The multiple phenotypic deficits in PKCgamma KO mice confound the interpretation of the proposed role of PKCgamma-expressing spinal neurons in neuropathic pain states. Additionally, the data show that the regulation of spinal dynorphin expression is a common critical feature of expression of neuropathic pain.

Enhanced evoked excitatory transmitter release in experimental neuropathy requires descending facilitation.

Nerve injury-induced afferent discharge is thought to elicit spinal sensitization and consequent abnormal pain. Experimental neuropathic pain, however, also depends on central changes, including descending facilitation arising from the rostral ventromedial medulla (RVM) and upregulation of spinal dynorphin. A possible intersection of these influences at the spinal level was explored by measuring evoked, excitatory transmitter release in tissues taken from nerve-injured animals with or without previous manipulation of descending modulatory systems. Spinal nerve ligation (SNL) produced expected tactile and thermal hyperesthesias. Capsaicin-evoked calcitonin gene-related peptide (CGRP) release was markedly enhanced in lumbar spinal tissue from SNL rats when compared with sham-operated controls. Enhanced, evoked CGRP release from SNL rats was blocked by anti-dynorphin A(1-13) antiserum; this treatment did not alter evoked release in tissues from sham-operated rats. Dorsolateral funiculus lesion (DLF) or destruction of RVM neurons expressing mu-opioid receptors with dermorphin-saporin, blocked tactile and thermal hypersensitivity, as well as SNL-induced upregulation of spinal dynorphin. Spinal tissues from these DLF-lesioned or dermorphin-saporin-treated SNL rats did not exhibit enhanced capsaicin-evoked CGRP-IR release. These data demonstrate exaggerated release of excitatory transmitter from primary afferents after injury to peripheral nerves, supporting the likely importance of increased afferent input as a driving force of neuropathic pain. The data also show that modulatory influences of descending facilitation are required for enhanced evoked transmitter release after nerve injury. Thus, convergence of descending modulation, spinal plasticity, and afferent drive in the nerve-injured state reveals a mechanism by which some aspects of nerve injury-induced hyperesthesias may occur.

Dynorphin-independent spinal cannabinoid antinociception.

Spinal antinociception produced by delta 9-tetrahydro-cannabinol (Delta(9)-THC) and other cannabinoid agonists has been suggested to be mediated by the release of dynorphin acting at the kappa opioid receptor. Alternatively, as cannabinoid receptors are distributed appropriately in the pain transmission pathway, cannabinoid agonists might act directly at the spinal level to inhibit nociception, without requiring dynorphin release. Here, these possibilities were explored using mice with a deletion of the gene encoding prodynorphin. Antinociceptive dose-response curves were constructed for spinal Delta(9)-THC and WIN 55,212-2 in prodynorphin knock-out mice and in wild-type littermates. WIN 55,212-2 and Delta(9)-THC were equipotent in the wild-type and prodynorphin knock-out mice. Spinal pretreatment with a kappa opioid receptor antagonist, nor-binaltorphimine (nor-BNI), did not alter the dose-response curves for either WIN 55,212-2 or Delta(9)-THC in prodynorphin knock-out and wild-type mice. However, the same dose of nor-BNI used blocked U50,488H-induced antinociception in both wild-type and prodynorphin knock-out mice, confirming kappa opioid receptor activity. Pretreatment with SR141716A, a cannabinoid receptor antagonist blocked the antinociceptive actions of both WIN 55,212-2 and Delta(9)-THC. These data support the conclusion that antinociception produced by spinal cannabinoids are likely to be mediated directly through activation of cannabinoid receptors without the requirement for dynorphin release or activation of kappa opioid receptors.

Pronociceptive effects of spinal dynorphin promote cannabinoid-induced pain and antinociceptive tolerance.

Recent studies indicate that sustained opioid administration produces increased expression of spinal dynorphin, which promotes enhanced sensitivity to non-noxious and noxious stimuli. Such increased "pain" may manifest behaviorally as a decrease in spinal antinociceptive potency. Here, the possibility of similar mechanisms in the antinociception of spinal cannabinoids was explored. Response thresholds to non-noxious mechanical and noxious thermal stimuli were assessed. Antinociception was determined using the 52 degrees C tail-flick test. Mice received repeated WIN 55,212-2, its inactive enantiomer, WIN 55,212-3 or vehicle (i.th., bid, 5 days). WIN 55,212-2, but not WIN 55,212-3 or vehicle, produced a time-related increased sensitivity to non-noxious and noxious stimuli. WIN 55,212-2, but not WIN 55,212-3 or vehicle, elicited a significant increase in lumbar spinal dynorphin content at treatment day 5. Increased sensitivity to mechanical and thermal stimuli produced by WIN 55,212-2 was reversed to baseline levels by i.th. MK-801 or dynorphin antiserum; control serum had no effect. WIN 55,212-2, but not WIN 55,212-3 or vehicle, produced dose-related antinociception and repeated administration resulted in antinociceptive tolerance. While MK-801 and dynorphin antiserum did not alter acute antinociception produced by WIN 55,212-2, these substances significantly blocked antinociceptive tolerance when given immediately prior to WIN 55,212-2 challenge on day 5. Daily MK-801 pretreatments, prior to WIN 55,212-2 injection, also produced a significant block of antinociceptive tolerance. These data suggest that like opioids, repeated spinal administration of a cannabinoid CB1 agonist elicits abnormal pain, which results in increased expression of spinal dynorphin. Manipulations that block cannabinoid-induced pain also block the behavioral manifestation of cannabinoid tolerance.

Inhibition of neuropathic pain by selective ablation of brainstem medullary cells expressing the mu-opioid receptor.

Neurons in the rostroventromedial medulla (RVM) project to spinal loci where the neurons inhibit or facilitate pain transmission. Abnormal activity of facilitatory processes may thus represent a mechanism of chronic pain. This possibility and the phenotype of RVM cells that might underlie experimental neuropathic pain were investigated. Cells expressing mu-opioid receptors were targeted with a single microinjection of saporin conjugated to the mu-opioid agonist dermorphin; unconjugated saporin and dermorphin were used as controls. RVM dermorphin-saporin, but not dermorphin or saporin, significantly decreased cells expressing mu-opioid receptor transcript. RVM dermorphin, saporin, or dermorphin-saporin did not change baseline hindpaw sensitivity to non-noxious or noxious stimuli. Spinal nerve ligation (SNL) injury in rats pretreated with RVM dermorphin-saporin failed to elicit the expected increase in sensitivity to non-noxious mechanical or noxious thermal stimuli applied to the paw. RVM dermorphin or saporin did not alter SNL-induced experimental pain, and no pretreatment affected the responses of sham-operated groups. This protective effect of dermorphin-saporin against SNL-induced pain was blocked by beta-funaltrexamine, a selective mu-opioid receptor antagonist, indicating specific interaction of dermorphin-saporin with the mu-opioid receptor. RVM microinjection of dermorphin-saporin, but not of dermorphin or saporin, in animals previously undergoing SNL showed a time-related reversal of the SNL-induced experimental pain to preinjury baseline levels. Thus, loss of RVM mu receptor-expressing cells both prevents and reverses experimental neuropathic pain. The data support the hypothesis that inappropriate tonic-descending facilitation may underlie some chronic pain states and offer new possibilities for the design of therapeutic strategies.

Pronociceptive actions of dynorphin maintain chronic neuropathic pain.

Whereas tissue injury increases spinal dynorphin expression, the functional relevance of this upregulation to persistent pain is unknown. Here, mice lacking the prodynorphin gene were studied for sensitivity to non-noxious and noxious stimuli, before and after induction of experimental neuropathic pain. Prodynorphin knock-out (KO) mice had normal responses to acute non-noxious stimuli and a mild increased sensitivity to some noxious stimuli. After spinal nerve ligation (SNL), both wild-type (WT) and KO mice demonstrated decreased thresholds to innocuous mechanical and to noxious thermal stimuli, indicating that dynorphin is not required for initiation of neuropathic pain. However, whereas neuropathic pain was sustained in WT mice, KO mice showed a return to baselines by post-SNL day 10. In WT mice, SNL upregulated lumbar dynorphin content on day 10, but not day 2, after injury. Intrathecal dynorphin antiserum reversed neuropathic pain in WT mice at post-SNL day 10 (when dynorphin was upregulated) but not on post-SNL day 2; intrathecal MK-801 reversed SNL-pain at both times. Opioid (mu, delta, and kappa) receptor density and G-protein activation were not different between WT and KO mice and were unchanged by SNL injury. The observations suggest (1) an early, dynorphin-independent phase of neuropathic pain and a later dynorphin-dependent stage, (2) that upregulated spinal dynorphin is pronociceptive and required for the maintenance of persistent neuropathic pain, and (3) that processes required for the initiation and the maintenance of the neuropathic pain state are distinct. Identification of mechanisms that maintain neuropathic pain appears important for strategies to treat neuropathic pain.

Dynorphin promotes abnormal pain and spinal opioid antinociceptive tolerance.

The nonopioid actions of spinal dynorphin may promote aspects of abnormal pain after nerve injury. Mechanistic similarities have been suggested between opioid tolerance and neuropathic pain. Here, the hypothesis that spinal dynorphin might mediate effects of sustained spinal opioids was explored. Possible abnormal pain and spinal antinociceptive tolerance were evaluated after intrathecal administration of [D-Ala(2), N-Me-Phe(4), Gly-ol(5)]enkephalin (DAMGO), an opioid mu agonist. Rats infused with DAMGO, but not saline, demonstrated tactile allodynia and thermal hyperalgesia of the hindpaws (during the DAMGO infusion) and a decrease in antinociceptive potency and efficacy of spinal opioids (tolerance), signs also characteristic of nerve injury. Spinal DAMGO elicited an increase in lumbar dynorphin content and a decrease in the mu receptor immunoreactivity in the spinal dorsal horn, signs also seen in the postnerve-injury state. Intrathecal administration of dynorphin A(1-17) antiserum blocked tactile allodynia and reversed thermal hyperalgesia to above baseline levels (i.e., antinociception). Spinal dynorphin antiserum, but not control serum, also reestablished the antinociceptive potency and efficacy of spinal morphine. Neither dynorphin antiserum nor control serum administration altered baseline non-noxious or noxious thresholds or affected the intrathecal morphine antinociceptive response in saline-infused rats. These data suggest that spinal dynorphin promotes abnormal pain and acts to reduce the antinociceptive efficacy of spinal opioids (i.e., tolerance). The data also identify a possible mechanism for previously unexplained clinical observations and offer a novel approach for the development of strategies that could improve the long-term use of opioids for pain.

Extraterritorial neuropathic pain correlates with multisegmental elevation of spinal dynorphin in nerve-injured rats.

Neuropathic pain is often associated with the appearance of pain in regions not related to the injured nerve. One mechanism that may underlie neuropathic pain is abnormal, spontaneous afferent drive which may contribute to NMDA-mediated central sensitization by the actions of glutamate and by the non-opioid actions of spinal dynorphin. In the present study, injuries to lumbar or sacral spinal nerves elicited elevation in spinal dynorphin content which correlated temporally and spatially with signs of neuropathic pain. The increase in spinal dynorphin content was coincident with the onset of tactile allodynia and thermal hyperalgesia. Injury to the lumbar (L(5)/L(6)) spinal nerves produced elevated spinal dynorphin content in the ipsilateral dorsal spinal quadrant at the L(5) and L(6) spinal segments and in the segments immediately adjacent. Lumbar nerve injury elicited ipsilateral tactile allodynia and thermal hyperalgesia of the hindpaw. In contrast, S(2) spinal nerve ligation elicited elevated dynorphin content in sacral spinal segments and bilaterally in the caudal lumbar spinal cord. The behavioral consequences of S(2) spinal nerve ligation were also bilateral, with tactile allodynia and thermal hyperalgesia seen in both hindpaws. Application of lidocaine to the site of S(2) ligation blocked thermal hyperalgesia and tactile allodynia of the hindpaws suggesting that afferent drive was critical to maintenance of the pain state. Spinal injection of antiserum to dynorphin A((1-17)) and of MK-801 both blocked thermal hyperalgesia, but not tactile allodynia, of the hindpaw after S(2) ligation. These data suggest that the elevated spinal dynorphin content consequent to peripheral nerve injury may drive sensitization of the spinal cord, in part through dynorphin acting directly or indirectly on the NMDA receptor complex. Furthermore, extrasegmental increases in spinal dynorphin content may partly underlie the development of extraterritorial neuropathic pain.

Amlodipine, a calcium channel inhibitor, and cocaine and ethanol's reinforcing effects.

The effects of amlodipine (from 0.1 to 3.0 mg/kg) on rats' pressing for rewarding brain stimulation, with and without cocaine administration, were assessed. None of the doses reliably modified the effects of cocaine. Also, amlodipine was given to two groups of rats taking alcohol: one group that was regularly taking a sweetened alcoholic beverage and the other taking an unsweetened alcoholic beverage. The only discernible effects of amlodipine on alcohol intake were associated with the highest dose and only with rats taking the sweetened beverage. The effects of this high dose could easily be attributable to behavioral toxicity elicited by the dose. In contrast, and confirming previous work, isradipine, another calcium channel inhibitor, produced reliable reductions on both cocaine's and alcohol's reinforcing effects. Despite the similarity of isradipine and amlodipine, isradipine apparently has some unique features with respect to cocaine and alcohol.

Isradipine combined with naltrexone persistently reduces the reward-relevant effects of cocaine and alcohol.

Previous studies have revealed that the combination of small doses of isradipine and naltrexone (ISR&NTX) blocks the ability of cocaine to enhance pressing for rewarding, lateral hypothalamic brain stimulation. Further, such combinations also reduce rats' intakes of alcoholic beverages. Here, we asked whether ISR&NTX would lose its ability to reduce the reinforcing effects of cocaine and alcohol when given daily. Specifically, after almost 2 months of daily injections, ISR&NTX blocked the expression of a cocaine-induced conditioned place preference (CPP). By themselves, ISR and NTX were not effective at blocking cocaine's effects. Subsequent to the CPP procedures, the rats continued to receive daily injections for another 3 weeks. During this time, they were given access to water and an alcoholic beverage for 2 h a day. As expected, placebo controls gradually increased their daily intakes until they were taking about 2 g/kg of ethanol daily. ISR, NTX, and ISR&NTX blocked the typical pattern of intakes. At the end of the 3-week period, the rats had received 80 consecutive daily injections. The data suggest that the salient effects of ISR&NTX do not wane. The data support the idea that ISR&NTX would be a useful pharmacotherapy for poly drug abuse.

Valproate reduces intake of alcoholic beverage among rats.

A series of experiments was carried out to assess the effects of valproate (VAL) on the intake of ethanol by rats. In Experiment 1, the effects of VAL (150 and 200 mg/kg, i.p.) were assessed across 10 days. Compared with controls, the 200 mg/kg dose reliably reduced intake of ethanol while also reliably increasing intake of water. The 150 mg/kg dose did not reliably reduce the intake of ethanol across the initial days, but it did across later days. Neither dose affected the total intake of fluids. Similarly, 5 days of oral dosing with VAL (400 and 600 mg/kg) reliably reduced the intake of ethanol without affecting the intake of water. However, body weights were reduced by the oral doses across the procedure. In another procedure, VAL (200 mg/kg, i.p.) produced a mild conditioned taste aversion to a saccharin solution, suggesting that VAL may reduce intake of ethanol because it produces a general malaise. However, this dose of VAL enhanced the intoxicating effects of ethanol (2.0 g/kg). Overall, the results are equivocal with respect to VAL as a potential medicine for treating alcohol misuse and alcoholism.

Combination of naltrexone and fluoxetine on rats' propensity to take alcoholic beverage.

Naltrexone (NTX) and fluoxetine (FLU) are useful for treating alcoholism and depression, respectively. Furthermore, these afflictions covary. Given the possibility that people might be prescribed NTX and FLU concurrently, we assessed the effects of these two agents on rats' propensity to drink an alcoholic beverage. Rats were given 65 days of access to a sweetened alcoholic beverage and water for 2 hr daily. At first, they took little ethanol, but after 20 days, they took on average 2.0 to 2.5 g/kg of ethanol, daily during the 2-hr session. They also took sufficient water to maintain their health. After 30 days, they were divided into four groups to receive, 30 min before the drinking session, 1 of 4 different kinds of injections. For the next 20 days, one group received placebo daily. Another group received 5 mg/kg of NTX daily and another 5 mg/kg of FLU daily. The fourth group received both 5 mg/kg of NTX and 5 mg/kg of FLU daily. After 20 days, the doses of NTX and FLU were doubled across an additional 10 days. Both NTX and FLU reduced rats' intake of alcoholic beverage. The combinations of NTX and FLU, however, were no more effective in reducing rats' intake of alcoholic beverage than either alone. Also, the small dose of NTX seemed to lose its effectiveness with repeated administrations. A second experiment confirmed the conclusion that small doses of NTX lose their effectiveness in suppressing intake of alcoholic beverage across repeated administrations. In summary, data provide no support for the idea that FLU and NTX would act synergistically to reduce propensity to take alcoholic beverages.

Isradipine and naltrexone in combination with isradipine interact with a period of abstinence to reduce rats' intakes of an alcoholic beverage.

Individually housed rats were placed on a daily regimen of only 2 hr a day to drink both water and a sweetened alcoholic beverage. Initially, rats took little ethanol, but after 3 weeks, they took, on average, >2.0 g/kg daily. With achievement of stable intakes, the rats were deprived of opportunity to drink ethanol for 24 days and then the daily regimen was reinstated. With the reinstatement, various injections were given daily for 25 days or more: placebos, doses of isradipine (1.0 or 3.0 mg/kg), naltrexone (3.0 mg/kg), and a combination of isradipine (1.0 mg/kg) and naltrexone (3.0 mg/kg). The combination produced favorable effects with the fewest limiting side-effects. The period of abstinence decreased daily intakes of ethanol and interacted with the drugs to produce large, sustained decreases in intakes of ethanol.

Periodic naltrexone and propensity to take alcoholic beverage.

For over 2 months, 45 rats were maintained on a daily regimen involving 2 hr a day of access to both water and palatable alcoholic beverage. At first, they took little ethanol. As days progressed, they eventually took over 2 g/kg of ethanol during the 2 hr. Previous research indicates that, without intervention, they would maintain this level of intake indefinitely. All rats were taken off the daily regimen for 30 days and then returned to it, i.e., rats received 30 days of "abstinence". For 35 days following abstinence, one-third of the subjects received placebos daily, one-third received naltrexone (NTX), 10 mg/kg, daily, and the one-third received NTX on days 1-5, 11-15, 21-25, and 31-35 and placebos on the other days. Abstinence reduced all rats' intakes of alcohol compared with pre-abstinence levels. Rats that received only placebos quickly returned to taking alcohol at pre-abstinence levels. Rats that received NTX daily increased their intakes up to the level normally expected for receiving NTX and no abstinence. Because rats receiving daily NTX always drank a fraction of the alcohol consumed by those receiving placebos, NTX's effects did not diminish. As rats sampled alcoholic beverage, however, the effects of abstinence did diminish. The rats of periodic NTX drank as rats getting NTX when they were given NTX and as rats getting placebos when they were given placebos. Furthermore, the rats of periodic NTX showed no carry-over effects from periods of NTX to no NTX. Abstinence and NTX together, apparently, reduce propensity to take alcoholic beverage more than either alone.

Naltrexone persistently reduces rats' intake of a palatable alcoholic beverage.

Rats were given 30 days of opportunity to take a sweetened alcoholic beverage and water for 2 hr/day. At first, they took little alcohol, but subsequently took, on average, 2.3 g/kg of alcohol/daily session. They also took sufficient water, during the 2-hr period, to maintain their health and to steadily gain weight. At the end of the 30 days, they were divided into four groups so that their intakes of alcohol were similar. All groups continued on the daily regimen, but each group received different injections. One group received placebos, whereas the other two groups received either 5.0 or 10.0 mg/kg, respectively, of naltrexone daily, 30 min before the drinking session. The fourth group received 5.0 mg/kg of naltrexone 12.5 hr before the session and another 5.0 mg/kg 30 min before the session. This regimen of dosing and daily opportunities to drink continued for 30 days. With the end of injections, subjects continued on the regimen for another 5 days. Naltrexone, dose-relatedly, reduced rats' intake of alcoholic beverage. Furthermore, with respect to reducing intake of alcohol, no tolerance or refractoriness were observed across the 30 days of dosing. Within a couple of days after dosing, levels of intake returned to predosing levels.