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Cognitive impairment in animal models of persistent pain
Abstracts
Paper Session 330: Nonhuman Animal Studies A. Molecular and Cellular Biology A02 - Mechanisms of Opioid Action (330) Spinal cord postsynaptic density-93 protein contributes to the development of opioid tolerance and dependence Y. Tao, W. Liaw, R. Johns, R. Petralia, D. Bredt; Johns Hopkins University School of Medicine, Baltimore, MD N-methyl-D-aspartate (NMDA) receptors regulate diverse functions including central sensitization during persistent pain and the development of opioid tolerance and dependence. Neuronal postsynaptic density-93 protein (PSD-93), a molecular scaffolding protein, binds to and clusters NMDA receptors at synapses, and modulates NMDA receptors synaptic function. We report here that spinal cord PSD-93 might be involved in the molecular mechanism of development of morphine tolerance and dependence. Reverse transcriptase-polymerase chain and immunoblot analyses reveal abundant expression of PSD-93 mRNA and protein in spinal dorsal horn. PSD-93 immunoreactivity is distributed mainly in the superficial dorsal horn of spinal cord. Under electron microcopy, immunogold-labeling for PSD-93 is associated with the postsynaptic membranes and co-localized with NMDA receptor subunit NR2A/2B at the synaptic sites in the superficial dorsal horn. Co-immunoprecipitation assay reveals interaction of PSD-93 with NR2A and NR2B in spinal cord fractions. Targeted disruption of PSD-93 gene not only reduces NMDA receptor-mediated excitatory postsynaptic potentials in the spinal dorsal horn neurons but also prevents the developments of NMDA receptor-dependent morphine tolerance and dependence. Moreover, knockdown of spinal cord PSD-93 attenuated the development of intrathecal morphine tolerance. Our results suggest that spinal cord PSD-93 may contribute to the molecular mechanism of development of opioid tolerance and dependence via modulation of synaptic NMDA receptors.
A06 - Regulation of Gene Expression (330) Quantitative trait locus (QTL) mapping of mechanical sensitivity and peripheral nerve injury-induced neuropathic mechanical allodynia in recombinant inbred (RI) mice using www.webqtl.org W. Lariviere, E. Chesler, S. Lee, J. Chung; University of Pittsburgh School of Medicine, Pittsburgh, PA Several efforts are currently in progress to discover new genes involved in pain sensitivity and the susceptibility to chronic pain. We present here a QTL mapping study using the online resource www.webqtl.org to determine genetic mechanisms directly from phenotypic information. Using a murine model of neuropathic pain (Kim and Chung model of constriction of L5 nerve distal to the ganglion), we surveyed the resulting mechanical allodynia out to 14 days after peripheral nerve injury (PNI) in 26 BXD RI strains of mice (204 mice in total). A large range of strain means of baseline mechanical thresholds to von Frey monofilament application and PNI-induced mechanical allodynia demonstrated the significant heritability of these traits. The strain means were correlated with genotypic variation across the genome using up to 779 markers available on www.webqtl.org. Interval mapping found 2 significant QTLs for baseline mechanical sensitivity on proximal chromosome (Chr.) 5 and distal Chr. 15. Three suggestive QTLs for PNI-induced mechanical allodynia at day 14 were found on Chr. 1, proximal Chr. 2, and distal Chr. 9. Using genetic correlation of WebQTL’s gene expression data and SNP analysis, positional and functional candidate genes were identified within the QTLs and will be tested. Strategies to analyze and confirm the suggestive QTLs will be presented. The findings will be compared to our previous genetic correlation analysis of these traits in standard inbred mouse strains. This study and previously published studies demonstrate that QTL mapping can be successfully performed of painrelevant traits in RI mouse strains. Future collaborative efforts centered on the use of BXD RI strains and www.webqtl.org will allow for the derivation of tissue-specific genetic information related simultaneously to a number of pain traits and non-pain-related traits listed on www.webqtl.org, genetic correlation analyses, and the determination of gene networks for pain phenotypes.
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A07 - Regulating Neuronal Excitability (330) Antihyperalgesic effect of herpes vector mediated knock-down of Nav1.7 sodium shannels in a rodent inflammatory model D. Yeomans, S. Levinson, W. Gilly, S. Wilson; Stanford University, Stanford, CA Nav1.7, a voltage-gated sodium channel is present in nociceptive neurons. Furthermore, levels of Nav1.7 change dramatically during some inflammatory pain states, implying a role in establishing and/or maintaining these pain states. Thus, the Nav1.7 channel may be a novel target for new approaches to the treatment of chronic pain. In order to establish the importance of this sodium channel in pain, we applied a recombinant herpes virus, encoding an antisense sequence for the Nav1.7 gene to the hindpaw skin of mice, with the goal of decreasing production of this channel in afferents innervating this skin area. The virus also encoded the gene for green fluorescent protein so that transfected cells could be readily identified. We then performed immunochemical, behavioral, and electrophysiological experiments to examine the effects of knock-down of this channel on pain processes related to inflammation. Application of the vector produced a clear decrease in immunohistochemical labeling for Nav1.7 and a decrease in the Nav1.7 sodium current in transfected neurons, and a dramatic decrease in inflammatory hyperalgesia in the treated area. These results indicate that Nav1.7 is a likely target for the development of new pharmaceutical tools for treating inflammatory pain. It may also be possible to use herpes vectors to inhibit production of endogenous Nav1.7 channels in inflammatory pain patients, thus providing long-term genetic therapy for their pain.
B. Systems (Physiology, Anatomy, Animal Models) B01 - Animal Models of Chronic Pain (330) Cognitive impairment in animal models of persistent pain V. Galhardo, M. Pais-Vieira; IBMC - University of Porto, Portugal Chronic pain is known to cause cognitive deficits in human subjects. These deficits range from attentional impairment to lower performance in memory recall tasks. However, few studies address similar issues in animal models of persistent pain. To understand the magnitude of deficits induced by persistent pain, we analysed several different cognitive domains. In this study we looked for spatial long-term memory, working memory and decision-making deficits that could be affected by persistent pain. Spatial long-term memory was evaluated by the water maze task, while the T-Maze was used to study working memory. For the decision-making evaluation we designed a new behavioral task where animals were trained to choose between one of two levers in order to gain a fixed reward. After a training period where both levers lead to equal rewards, the conditions where changed without notice: one lever dispensed a large reward in 3 out of 10 trials and no reward in the others; the second lever dispensed a regular reward in 7 out of 10 trials. Decision between the two levers was monitored for 120 trials. Comparison between control and CFA-induced monoarthritic animals revealed that monoarthritic animals needed several more trials to reach levels of performance equal to controls, although after fully trained they present no spatial memory deficit. In the T-maze monoarthritic animals were not able to reach the basic criterion of 85% of correct choices needed for apply the working memory test. On the decision making task, monoarthritic animals have persistently chosen the lever with less net gain (i.e. with large reward), while controls chose to prefer reliable small rewards over scarce large rewards. Taken together these results show specific cognitive deficits induced by persistent pain conditions which have impact not only on memory acquisition, but also in long-term planning. Support: FCT (POCTI/NSE/38995/2001), EU (IST-2001-34892).
Paper Session 330: Nonhuman Animal Studies A. Molecular and Cellular Biology A02 - Mechanisms of Opioid Action (330) Spinal cord postsynaptic density-93 protein contributes to the development of opioid tolerance and dependence Y. Tao, W. Liaw, R. Johns, R. Petralia, D. Bredt; Johns Hopkins University School of Medicine, Baltimore, MD N-methyl-D-aspartate (NMDA) receptors regulate diverse functions including central sensitization during persistent pain and the development of opioid tolerance and dependence. Neuronal postsynaptic density-93 protein (PSD-93), a molecular scaffolding protein, binds to and clusters NMDA receptors at synapses, and modulates NMDA receptors synaptic function. We report here that spinal cord PSD-93 might be involved in the molecular mechanism of development of morphine tolerance and dependence. Reverse transcriptase-polymerase chain and immunoblot analyses reveal abundant expression of PSD-93 mRNA and protein in spinal dorsal horn. PSD-93 immunoreactivity is distributed mainly in the superficial dorsal horn of spinal cord. Under electron microcopy, immunogold-labeling for PSD-93 is associated with the postsynaptic membranes and co-localized with NMDA receptor subunit NR2A/2B at the synaptic sites in the superficial dorsal horn. Co-immunoprecipitation assay reveals interaction of PSD-93 with NR2A and NR2B in spinal cord fractions. Targeted disruption of PSD-93 gene not only reduces NMDA receptor-mediated excitatory postsynaptic potentials in the spinal dorsal horn neurons but also prevents the developments of NMDA receptor-dependent morphine tolerance and dependence. Moreover, knockdown of spinal cord PSD-93 attenuated the development of intrathecal morphine tolerance. Our results suggest that spinal cord PSD-93 may contribute to the molecular mechanism of development of opioid tolerance and dependence via modulation of synaptic NMDA receptors.
A06 - Regulation of Gene Expression (330) Quantitative trait locus (QTL) mapping of mechanical sensitivity and peripheral nerve injury-induced neuropathic mechanical allodynia in recombinant inbred (RI) mice using www.webqtl.org W. Lariviere, E. Chesler, S. Lee, J. Chung; University of Pittsburgh School of Medicine, Pittsburgh, PA Several efforts are currently in progress to discover new genes involved in pain sensitivity and the susceptibility to chronic pain. We present here a QTL mapping study using the online resource www.webqtl.org to determine genetic mechanisms directly from phenotypic information. Using a murine model of neuropathic pain (Kim and Chung model of constriction of L5 nerve distal to the ganglion), we surveyed the resulting mechanical allodynia out to 14 days after peripheral nerve injury (PNI) in 26 BXD RI strains of mice (204 mice in total). A large range of strain means of baseline mechanical thresholds to von Frey monofilament application and PNI-induced mechanical allodynia demonstrated the significant heritability of these traits. The strain means were correlated with genotypic variation across the genome using up to 779 markers available on www.webqtl.org. Interval mapping found 2 significant QTLs for baseline mechanical sensitivity on proximal chromosome (Chr.) 5 and distal Chr. 15. Three suggestive QTLs for PNI-induced mechanical allodynia at day 14 were found on Chr. 1, proximal Chr. 2, and distal Chr. 9. Using genetic correlation of WebQTL’s gene expression data and SNP analysis, positional and functional candidate genes were identified within the QTLs and will be tested. Strategies to analyze and confirm the suggestive QTLs will be presented. The findings will be compared to our previous genetic correlation analysis of these traits in standard inbred mouse strains. This study and previously published studies demonstrate that QTL mapping can be successfully performed of painrelevant traits in RI mouse strains. Future collaborative efforts centered on the use of BXD RI strains and www.webqtl.org will allow for the derivation of tissue-specific genetic information related simultaneously to a number of pain traits and non-pain-related traits listed on www.webqtl.org, genetic correlation analyses, and the determination of gene networks for pain phenotypes.
S7
A07 - Regulating Neuronal Excitability (330) Antihyperalgesic effect of herpes vector mediated knock-down of Nav1.7 sodium shannels in a rodent inflammatory model D. Yeomans, S. Levinson, W. Gilly, S. Wilson; Stanford University, Stanford, CA Nav1.7, a voltage-gated sodium channel is present in nociceptive neurons. Furthermore, levels of Nav1.7 change dramatically during some inflammatory pain states, implying a role in establishing and/or maintaining these pain states. Thus, the Nav1.7 channel may be a novel target for new approaches to the treatment of chronic pain. In order to establish the importance of this sodium channel in pain, we applied a recombinant herpes virus, encoding an antisense sequence for the Nav1.7 gene to the hindpaw skin of mice, with the goal of decreasing production of this channel in afferents innervating this skin area. The virus also encoded the gene for green fluorescent protein so that transfected cells could be readily identified. We then performed immunochemical, behavioral, and electrophysiological experiments to examine the effects of knock-down of this channel on pain processes related to inflammation. Application of the vector produced a clear decrease in immunohistochemical labeling for Nav1.7 and a decrease in the Nav1.7 sodium current in transfected neurons, and a dramatic decrease in inflammatory hyperalgesia in the treated area. These results indicate that Nav1.7 is a likely target for the development of new pharmaceutical tools for treating inflammatory pain. It may also be possible to use herpes vectors to inhibit production of endogenous Nav1.7 channels in inflammatory pain patients, thus providing long-term genetic therapy for their pain.
B. Systems (Physiology, Anatomy, Animal Models) B01 - Animal Models of Chronic Pain (330) Cognitive impairment in animal models of persistent pain V. Galhardo, M. Pais-Vieira; IBMC - University of Porto, Portugal Chronic pain is known to cause cognitive deficits in human subjects. These deficits range from attentional impairment to lower performance in memory recall tasks. However, few studies address similar issues in animal models of persistent pain. To understand the magnitude of deficits induced by persistent pain, we analysed several different cognitive domains. In this study we looked for spatial long-term memory, working memory and decision-making deficits that could be affected by persistent pain. Spatial long-term memory was evaluated by the water maze task, while the T-Maze was used to study working memory. For the decision-making evaluation we designed a new behavioral task where animals were trained to choose between one of two levers in order to gain a fixed reward. After a training period where both levers lead to equal rewards, the conditions where changed without notice: one lever dispensed a large reward in 3 out of 10 trials and no reward in the others; the second lever dispensed a regular reward in 7 out of 10 trials. Decision between the two levers was monitored for 120 trials. Comparison between control and CFA-induced monoarthritic animals revealed that monoarthritic animals needed several more trials to reach levels of performance equal to controls, although after fully trained they present no spatial memory deficit. In the T-maze monoarthritic animals were not able to reach the basic criterion of 85% of correct choices needed for apply the working memory test. On the decision making task, monoarthritic animals have persistently chosen the lever with less net gain (i.e. with large reward), while controls chose to prefer reliable small rewards over scarce large rewards. Taken together these results show specific cognitive deficits induced by persistent pain conditions which have impact not only on memory acquisition, but also in long-term planning. Support: FCT (POCTI/NSE/38995/2001), EU (IST-2001-34892).