Peripheral nerve injury promotes morphine-seeking behavior in rats during extinction.

Chronic neuropathic pain and prescription opioid abuse represent highly interconnected societal problems. We used a rat model of spared nerve injury (SNI) and an intravenous drug self-administration paradigm to investigate the impact of a neuropathic pain state on morphine-seeking behavior in extinction (i.e. when morphine is withheld). SNI, sham-operated and naive groups exhibited similar levels of active lever presses for morphine infusions on a fixed ratio 1 (FR1) schedule. Self-administration of morphine, but not vehicle, attenuated nerve injury-induced mechanical allodynia in SNI rats. Under these same conditions, mechanical paw withdrawal thresholds in sham-operated and naive groups were largely unaltered. However, SNI rats showed higher levels of morphine-seeking behavior compared to sham-operated or naïve groups in extinction (i.e. when vehicle was substituted for morphine). Interestingly, the perseveration of morphine-seeking behavior observed during extinction was only present in the SNI group despite the fact that all groups had a similar history of morphine self-administration intake. Our results suggest that different motivational states associated with neuropathic pain promote morphine-seeking behavior in extinction. Drug self-administration paradigms may be useful for evaluating analgesic efficacy and motivational properties associated with opioid reinforcers in pathological pain states.

Inflammatory and Neuropathic Nociception is Preserved in GPR55 Knockout Mice.

The G-protein coupled receptor GPR55 has been postulated to serve as a novel cannabinoid receptor. A previous report indicated that GPR55 knockout mice fail to develop mechanical hyperalgesia, suggesting a pro-nociceptive role for GPR55 in the control of nociceptive responding. However, GPR55 knockout mice remain incompletely characterized in models of pathological pain. Here we provide a comprehensive assessment of responses of GPR55 knockout and wild-type mice to mechanical and thermal (heat, cold) stimulation in multiple, mechanistically distinct models of inflammatory and neuropathic pain. Inflammatory sensitization was produced by intraplantar administration of capsaicin, formalin or complete Freund's adjuvant. No differences in responding were detected between GPR55 knockout and wild-type mice in any model of inflammatory nociception assessed. Neuropathic pain was induced by partial sciatic nerve ligation (which induces hypersensitivity to mechanical, cold and heat stimulation) or by treatment with the chemotherapeutic agent paclitaxel (which induces hypersensitivity to mechanical and cold stimulation only). No differences were observed between GPR55 knockout and wild type mice in either development or maintenance of neuropathic nociception in either neuropathic pain model. In conclusion, genetic deletion of GPR55 did not alter the development of pathological pain in adult mice in any chronic pain model evaluated.

Small molecule inhibitors of PSD95-nNOS protein-protein interactions suppress formalin-evoked Fos protein expression and nociceptive behavior in rats.

Excessive activation of NMDA receptor (NMDAR) signaling within the spinal dorsal horn contributes to central sensitization and the induction and maintenance of pathological pain states. However, direct antagonism of NMDARs produces undesirable side effects which limit their clinical use. NMDAR activation produces central sensitization, in part, by initiating a signaling cascade that activates the enzyme neuronal nitric oxide synthase (nNOS) and generates the signaling molecule nitric oxide. NMDAR-mediated activation of nNOS requires a scaffolding protein, postsynaptic density protein 95kDa (PSD95), which tethers nNOS to NMDARs. Thus, disrupting the protein-protein interaction between PSD95 and nNOS may inhibit pro-nociceptive signaling mechanisms downstream of NMDARs and suppress central sensitization while sparing unwanted side effects associated with NMDAR antagonists. We examined the impact of small molecule PSD95-nNOS protein-protein interaction inhibitors (ZL006, IC87201) on both nociceptive behavior and formalin-evoked Fos protein expression within the lumbar spinal cord of rats. Comparisons were made with ZL007, an inactive analog of ZL006, and the NMDAR antagonist MK-801. IC87201 and ZL006, but not ZL007, suppressed phase 2 of formalin-evoked pain behavior and decreased the number of formalin-induced Fos-like immunoreactive cells in spinal dorsal horn regions associated with nociceptive processing. MK-801 suppressed Fos protein expression in both dorsal and ventral horns. MK-801 produced motor ataxia in the rotarod test whereas IC87201 and ZL006 failed to do so. ZL006 but not ZL007 suppressed paclitaxel-induced mechanical and cold allodynia in a model of chemotherapy-induced neuropathic pain. Co-immunoprecipitation experiments revealed the presence of the PSD95-nNOS complex in lumbar spinal cord of paclitaxel-treated rats, although ZL006 did not reliably disrupt the complex in all subjects. The present findings validate use of putative small molecule PSD95-nNOS protein-protein interaction inhibitors as novel analgesics and demonstrate, for the first time, that these inhibitors suppress inflammation-evoked neuronal activation at the level of the spinal dorsal horn.

Self-medication of a cannabinoid CB2 agonist in an animal model of neuropathic pain.

Drug self-administration methods were used to test the hypothesis that rats would self-medicate with a cannabinoid CB(2) agonist to attenuate a neuropathic pain state. Self-medication of the CB(2) agonist (R,S)-AM1241, but not vehicle, attenuated mechanical hypersensitivity produced by spared nerve injury. Switching rats from (R,S)-AM1241 to vehicle self-administration also decreased lever responding in an extinction paradigm. (R,S)-AM1241 self-administration did not alter paw withdrawal thresholds in sham-operated or naive animals. The percentage of active lever responding was similar in naive groups self-administering vehicle or (R,S)-AM1241. The CB(2) antagonist SR144528 blocked both antiallodynic effects of (R,S)-AM1241 self-medication and the percentage of active lever responding in neuropathic (but not naive) rats. Neuropathic and sham groups exhibited similar percentages of active lever responding for (R,S)-AM1241 on a fixed ratio 1 (FR1) schedule. However, neuropathic animals worked harder than shams to obtain (R,S)-AM1241 when the schedule of reinforcement was increased (to FR6). (R,S)-AM1241 self-medication on FR1, FR3, or FR6 schedules attenuated nerve injury-induced mechanical allodynia. (R,S)-AM1241 (900µg intravenously) failed to produce motor ataxia observed after administration of the mixed CB(1)/CB(2) agonist WIN55,212-2 (0.5mg/kg intravenously). Our results suggest that cannabinoid CB(2) agonists may be exploited to treat neuropathic pain with limited drug abuse liability and central nervous system side effects. These studies validate the use of drug self-administration methods for identifying nonpsychotropic analgesics possessing limited abuse potential. These methods offer potential to elucidate novel analgesics that suppress spontaneous neuropathic pain that is not measured by traditional assessments of evoked pain.

Unmasking the tonic-aversive state in neuropathic pain.

Tonic pain has been difficult to demonstrate in animals. Because relief of pain is rewarding, analgesic agents that are not rewarding in the absence of pain should become rewarding only when there is ongoing pain. We used conditioned place preference to concomitantly determine the presence of tonic pain in rats and the efficacy of agents that relieve it. This provides a new approach for investigating tonic pain in animals and for evaluating the analgesic effects of drugs.

Endocannabinoids at the spinal level regulate, but do not mediate, nonopioid stress-induced analgesia.

Recent work in our laboratories has demonstrated that an opioid-independent form of stress-induced analgesia (SIA) is mediated by endogenous cannabinoids [Hohmann et al., 2005. Nature 435, 1108]. Non-opioid SIA, induced by a 3-min continuous foot shock, is characterized by the mobilization of two endocannabinoid lipids--2-arachidonoylglycerol (2-AG) and anandamide--in the midbrain periaqueductal gray (PAG). The present studies were conducted to examine the contributions of spinal endocannabinoids to nonopioid SIA. Time-dependent increases in levels of 2-AG, but not anandamide, were observed in lumbar spinal cord extracts derived from shocked relative to non-shocked rats. Notably, 2-AG accumulation was of smaller magnitude than that observed previously in the dorsal midbrain following foot shock. 2-AG is preferentially degraded by monoacylglycerol lipase (MGL), whereas anandamide is hydrolyzed primarily by fatty-acid amide hydrolase (FAAH). This metabolic segregation enabled us to manipulate endocannabinoid tone at the spinal level to further evaluate the roles of 2-AG and anandamide in nonopioid SIA. Intrathecal administration of the competitive CB1 antagonist SR141716A (rimonabant) failed to suppress nonopioid SIA, suggesting that supraspinal rather than spinal CB1 receptor activation plays a pivotal role in endocannabinoid-mediated SIA. By contrast, spinal inhibition of MGL using URB602, which selectively inhibits 2-AG hydrolysis in the PAG, enhanced SIA through a CB1-selective mechanism. Spinal inhibition of FAAH, with either URB597 or arachidonoyl serotonin (AA-5-HT), also enhanced SIA through a CB1-mediated mechanism, presumably by increasing accumulation of tonically released anandamide. Our results suggest that endocannabinoids in the spinal cord regulate, but do not mediate, nonopioid SIA.

Inhibition of fatty-acid amide hydrolase enhances cannabinoid stress-induced analgesia: sites of action in the dorsolateral periaqueductal gray and rostral ventromedial medulla.

Recent research in our laboratory has demonstrated that stress activates an endogenous cannabinoid mechanism that suppresses sensitivity to pain [Nature 435 (2005) 1108]. In this work, CB(1) antagonists administered systemically blocked stress-induced analgesia induced by brief, continuous foot-shock. The present studies were conducted to examine the role of cannabinoid CB(1) receptors in the brainstem rostral ventromedial medulla (RVM) and midbrain dorsolateral periaqueductal gray (PAG) in cannabinoid stress-induced analgesia (SIA). Pharmacological blockade of vanilloid TRPV1 receptors with capsazepine, administered systemically, did not alter cannabinoid SIA, suggesting that cannabinoid SIA was not dependent upon TRPV1. Microinjection of the competitive CB(1) antagonist rimonabant (SR141716A) into either the RVM or dorsolateral PAG suppressed stress antinociception in this model. Rimonabant was maximally effective following microinjection into the dorsolateral PAG. The fatty-acid amide hydrolase (FAAH) inhibitor arachidonoyl serotonin (AA-5-HT) was subsequently used to block hydrolysis of endocannabinoids and enhance SIA. Systemic and site-specific injections of AA-5-HT into either the dorsolateral PAG or RVM induced CB(1)-mediated enhancements of SIA. Palmitoyltrifluoromethylketone, a potent inhibitor of FAAH and phospholipase A2 activity, administered systemically, exerted similar effects. In all conditions, the antinociceptive effects of each FAAH inhibitor were completely blocked by coadministration of the CB(1) antagonist rimonabant. The present results provide evidence that a descending cannabinergic neural system is activated by environmental stressors to modulate pain sensitivity in a CB(1)-dependent manner.

Modeling a task that is sensitive to dementia of the Alzheimer's type: individual differences in acquisition of a visuo-spatial paired-associate learning task in rhesus monkeys.

Early detection of progressive diseases such as Alzheimer's Disease (AD) is crucial for both the treatment and study of the disease. Performance on a visuo-spatial paired-associates learning (vsPAL) task was recently shown to reliably predict a diagnosis of AD in aged populations. The present study reports the development of this vsPAL task for use in nonhuman primates. Translation of vsPAL to a nonhuman model may provide improved preclinical tools for study of the etiology and treatment of dementia. Twelve young adult male rhesus monkeys were trained to perform the vsPAL task concurrently with tests comprising a nonhuman primate neuropsychological test battery. Monkeys successfully learned to perform vsPAL and did so in a task-difficulty ranked fashion. Despite significant individual differences in capability in the acquisition of the recognition memory aspects of the task, all monkeys evidenced the ability to learn within-trial, i.e. to improve with repeated stimulus-location pairings. These results support the use of vsPAL performance under various challenge conditions to investigate the possible substrates of early cognitive decline in AD. Comparison of performance on vsPAL with performance on other memory tasks in the battery will be of more general use in differentiating mechanisms involved in various aspects of mnemonic function.

Ketamine impairs multiple cognitive domains in rhesus monkeys.

Available evidence suggests that recreational use and abuse of the dissociative anaesthetic ketamine is increasing. Characterization of the cognitive risks of ketamine exposure contributes substantially to understanding this growing public health threat. Although prior human studies demonstrate that ketamine impairs a range of cognitive skills, investigation in nonhuman models permits more precise exploration of neurochemical mechanisms which may underlie detrimental behavioral effects. Adult male rhesus monkeys (N=7) were trained on a neuropsychological battery including tests of memory (delayed match-to-sample, DMS; self-ordered spatial search, SOSS), reaction time (RT), reinforcer efficacy and sustained attention (progressive ratio, PR) and fine motor coordination (bimanual motor skill, BMS). Battery performance was then serially challenged with acute doses of ketamine (0.3, 1.0, 1.78 mg/kg IM). Ketamine impaired DMS and SOSS in a dose x difficulty dependent manner with the most difficult task conditions disrupted at the 1.0 and 1.78 mg/kg doses. Thus, both visual recognition memory and working memory indices were affected. Ketamine also slowed RT and BMS performance and interfered with PR performance at the 1.78 mg/kg dose. Overall the present findings confirm that ketamine interferes with multiple aspects of cognition at subanesthetic doses in monkeys.

Differential muscarinic and NMDA contributions to visuo-spatial paired-associate learning in rhesus monkeys.

RATIONALE: Early, accurate detection of degenerative neurological disorders such as Alzheimer's disease (AD) is essential for therapies designed to slow disease progression. Performance of a touch-screen mediated visuo-spatial paired-associates learning (vsPAL) task predicts neurocognitive decline in elderly populations presenting with mild cognitive impairment and distinguishes AD patients from elderly depressed individuals. Translation of this cognitive task to a non-human model may therefore provide an improved tool for study of the etiology and treatment of dementia. OBJECTIVE: The goal of the current study was to contrast cholinergic and glutamatergic contributions to performance of this AD-sensitive task by challenging rhesus monkeys performing vsPAL with muscarinic antagonist and non-competitive NMDA antagonist drugs. METHODS: Seven monkeys were trained to perform vsPAL and then serially challenged with acute doses of scopolamine (3, 10, 17 microg/kg, IM) and ketamine (0.3, 1.0, 1.78 mg/kg, IM). RESULTS: Scopolamine produced a dosexdifficulty related impairment of both recognition memory and incremental acquisition aspects of task performance. In contrast, ketamine administration resulted in a dose-dependent impairment of recognition memory but not incremental acquisition. CONCLUSIONS: Monkeys' performance of a task sensitive to AD in humans was impaired by two classic pharmacological models of cognitive impairment, therefore supporting the use of this nonhuman model to explore mechanisms of AD-associated cognitive decline. The differential pattern of impairment observed is consistent with a hypothesis that muscarinic mechanisms are required for linking external events with an existing internal representation, whereas NMDA mechanisms are required for the formation/strengthening of such an internal representation.