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Regulation of Gene Expression

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ANTIHYPERALGESIA INDUCED BY EXPOSURE OF MOUSE SKIN TO A HERPES SIMPLEX VIRUS WHICH ENCODES ANTISENSE FOR CGRP

Lu, Y.*1, Wilson, S.P.3, Laurito, C. E.2, and Yeomans. D. C.1, 2 Depts. of Anatomy and Cell Biology and Anesthesiology2, Univ of Illinois at Chicago, Chicago, IL 60612; Dept. of Pharmacology, Univ of South Carolina School of Medicine, Columbia, SC, 29208

Aim of Investigation: Calcitonin gene related peptide (CGRP) is released from primary afferent terminals upon strong noxious stimulation, and has been demonstrated to be critical in the development of some kinds of centrally-mediated hyperalgesia. In this study, attenuated herpes vectors were used to insert the gene for the antisense to CGRP mRNA into primary nociceptive afferents to determine whether blockade of CGRP synthesis would attenuate induced hyperalgesia in mice.

Methods: The cDNA for rat CGRP was inserted into a shuttle plasmid in reverse orientation and inserted into the thymidine ki-nase sequence of a herpes simplex type I virus (KantiCGRP). A virus (KHZ) expressing the bacterial marker peptide gene lac-Z was constructed as a control. The dorsal hindpaw skin of female Swiss-Webster mice was scarified and topically treated with vehicle or a solution containing either KantiCGRP or KHZ (approx 8 X 10s Pfu). Baseline foot withdrawal latencies evoked by a C fiber-selective low rate of heating were measured biweekly after treatment. C fiber nociceptors innervating the infected skin area were then sensitized with topical capsaicin, following which response latencies were again measured and compared among the 3 groups.

Results: Mice exposed to KHZ or vehicle demonstrated profound capsaicin-induced sensitization of the foot withdrawal responses. Conversely, in mice infected with KantiCGRP sensitization was reduced or eiiminated for up to 17 weeks after infection. Conclusion: Introduction of CGRP mRNA antisense into nociceptive afferents using a herpes vector produces antihyperalgesia, presumably mediated by the inhibition of the synthesis and consequent release of endogenous CGRP. Further experiments will determine whether correlative decreases in message and peptide occur. Acknowledgments: This study was supported by USPHS Grant DA08256 (DCY); NSF Grant IBN-9728736 (SPW).

STIMULUS-DEPENDENT ANALGESIA AFTER INFECTION OF PRIMATE SKIN WITH A TRANSGENIC HERPES VIRUS ENCODING HUMAN PROENKEPHALIN

D. C. Yeomans1, 2. Y. Lu*, C. Laurito2, P. Kirn*, E. G. Votta-Velis*2, and S. Wilson3 Depts. of Anatomy and Cell Biology and Anesthesiology, Univ. of Illinois at Chicago, Chicago, Illinois, 60612, USA and ^Dept. of Pharmacology, Univ. of South Carolina, Columbia, SC, 29208, USA.

Aim of Investigation: The present study was designed to 1) establish a behavioral nociceptimetric model in macaque monkeys that allows for differential assessment of responses mediated by the activation ofA6 or C fiber nociceptors; and 2) determine whether cutaneous application of a human proenkephalin (HPE) encoding herpes virus attenuates nociceptive responses to heat and to cutaneous application of capsaicin.

Methods: Six stump-tailed macaques were lightly anesthetized with Propofol IV. 1) Based on our rodent work, we tested the dose- dependent effects of IV morphine and topical capsaicin on withdrawal latencies evoked by high or low rate heating of hairy hind paw skin to assess responses to A6 or C fiber nociceptor activation respectively. 2) We also determined the effects of topical application of a HPE encoding virus (KHPE) or a control virus which encoded the marker gene lac Z (KHZ) on basal foot with drawal latencies and latencies after capsaicin application, and determined whether these effects were naloxone reversible.

Results: Statistical analysis revealed that morphine was significantly more potent in attenuating responses to low vs. high rate heating, and that capsaicin decreased low rather than high rate response latencies. Both of these results indicate that high rate responses are mediated by A5 activation, whereas low rate heating evokes responses mediated by C nociceptors. In addition, our results indicate that application of KHPE, but not KHZ produced a localized, long duration anti-hyperalgesic effect, an effect which was partially blocked by intrathecal naloxone.

Conclusion: These results indicate that heating primate hairy skin at different rates allows for separate assessment ofC or A mediated responses, and that insertion of HPE gene into primary afferents using a viral vector may be a viable means of attenuating hyperalgesia in primates. Acknowledgments: DA08256 (DCY); IBN-9728736 (SPW).

NEURONAL SPECIFIC AND NGF INDUCIBLE EXPRESSION DIRECTED BY THE PREPROTACHYKININ-A PROMOTER DELIVERED BY AN ADENO-ASSOCIATED VIRAL VECTOR

Patrick T. Harrison*, Robert G. Dalziel*, Lesley Gerrard*, and John P. Ouinn* (SPON: C. HICK) Dept of Veterinary Pathology, The Univ of Edinburgh, Edinburgh EH9 1QH

Aim of Investigation: Generation of virus vectors for manipulation of sensory neuronal phenotype. Use a preprotachykinin-A (PPT) promoter fragment to support neuronal restricted expression of a transgene packaged as an adeno-associated virus (AAV) particle. Expression from this cassette would persist for at least several months.

Methods: The rat PPT promoter fragment spanning -865 to +92 was cloned upstream of the luciferase reporter gene and inserted between the packaging signals for AAV. Using a packaging protocol, virions were generated that contained only the PPT/luciferase transgene. Virus particles were analysed in primary cultures of rat dorsal root ganglion (DRG) for expression of luciferase. Results: The virions exhibit neuronal specific expression of the transgene. Luciferase activity is detected from 7-28 days in culture. Longer time points were not done. This PPT promoter for the first time was demonstrated to be NGF inducible (no NGF stimulated expression of this promoter region is observed when microin-jected).

Conclusions: These viruses can potentially be used to manipulate sensory neuronal expression in vivo by replacing luciferase with transgene of choice or an anti-sense molecule. Expression is considerably longer than plasmid DNA alone. The stimulation of promoter activity by NGF also allows a model to rapidly dissect signal transduction pathways in vivo. Acknowledgments: Wellcome Trust for support

P-ENDORPHIN EXPRESSION BY A REPLICATION-DEFECTIVE ADENO-ASSOCIATED VIRUS

A.JMannes. Robert M Caudle2, Zoltan Olah2*, Alan A. Finegold2, Ph.D.*, Guang-Pmg Gao, Ph.D.3, Michael J. ladarola2, Ph.D. Univ of Pennsylvania, Dept of Anesthesia, 3400 Spruce St, Philadelphia, PANIDR/NIHHGT

Aims of Investigation. Gene therapy may offer an alternative treatment of the severe, intractable pain often accompanying the terminal stages of cancer Previous work with a replication-defective adenovirus expressing the endogenous opioid, beta-endorphm, demonstrated anaigesia in a rat pain model. Although the virus reversed heat hyperalgesia, the effects were transient To prolong expression and resulting analgesic effects, the same fusion gene was cloned into the adeno-associated viral backbone. The present study was designed to show that cultured cells transduced with the virus would express extracellular P-endorphin. Methods. A replication-defective adeno-associated virus (AAV) was made containing the CMV promoter driving expression of a fusion between a prepro-NGF leader sequence and human (3-endorphm1 (AAVNGFpEND) Cells (cell line 8431-containing the adenoviral El and E4 genes) were grown to confluence in 12-well plates and AAVNGFpEND was added at day 0 (100, 1000 and 10,000 particles/cell) and the media sampled at days 0, 1 and 3 (3-endorphm was quantified by immunoassay in triplicate and reported in pmoles/ml.

Results: Analysis of the samples demonstrated high levels of p-endorphm expression in vitro in both a time- and dose-dependent response. A maximal response of 1 3 pmoles/ml media in cultured cells (3 days, 10,000 particles/cell) represents 40-fold increases as compared to p-endorphin CSF levels found in rat (35 fmole/ml) or human (15 fmole/ml)

Conclusions. In vitro transfection with AAVNGFpEND showed significant expression of extracellular p-endorphin The maximal response is 50% less than the expression with the maximal adenoviral vector but is still at a physiologically significant levels This demonstrates the feasibility of this approach for future work in rat pain models to assess prolonged behavioral changes in pain sensitivity.

References 1) Brain Research 272 75-85, 1988, 2) J Neurochem-istry 64:475-481, 1995

MET-ENKEPHALIN IS PREFERENTIALLY TRANSPORTED TO THE PERIPHERAL PROCESSES OF PRIMARY AFFERENT FIBRES IN BOTH NORMAL AND PROENKEPHALIN A OVEREXPRESSING RATS

JM Antunes Bras1*, MC Lombard2, F Cessehn5, M Hamon and M. Pohl. INSERM U288, Faculte de Medecme Pitie-Salpetnere, 75634 Pans Cedex 13, France, INSERM U161, 2 rue d'Alesia, 75014 Pans, France

Aim of Investigation- We previously demonstrated that the prepro-enkephalin A (pEnkA) gene is expressed in pnmary afferent neurons (PAN) of the rat Synthesis and accumulation ofmet-enkephalm (ME) in PAN, and transport of the peptide to their central and/or peripheral processes, were examined in control rats and in animals overexpressmg ME.

Methods. Application ofnon-replicative recombmant Herpes simplex virus-1 beanng the rat preproenkephalin A cDNA (HSVLat Enk;) on a scanfied footpad surface was used for dnvmg ME over-expression in dorsal root ganglia (DRG) cells. In control rats and in those infected by HSVLat Enk;, sciatic nerve or dorsal roots of lumbar (L4, L5) DRG were exposed and tightly ligatured on one side Tissues were processed 24 h later for immunohistochemical labeling of ME.

Results: In intact animals, DRG neuronal cell bodies containing immunoreactive ME-like matenal (MELM) were scarce and only few neuronal processes immunolabeled for ME were seen at the penpheral output of the DRG. Nerves ligature (sciatic or dorsal roots) produced an accumulation of MELM only in the sciatic nerve. In HSVLat Enk^-mfected animals, numerous neuronal cell bodies and fibres were labeled by anti-ME antibodies in DRG In addition, MELM accumulated heavily at the proximal side of ligatured sciatic nerve, and a few ME immunoreactive nerve fibres were observed in ligatured dorsal roots.

Conclusion- Thanks to the transfer of the pEnkA cDNA to rat DRG neurons, which resulted in a important production and accumulation of ME, the transport of the peptide (rather difficult to be evidenced in control animals) was shown to be preferentially (but not exclusively) directed to the penpheral processes of PAN. Our results support the existence of a neuronal source of penpheral MEE

ACTIVITY-DEPENDENT ACTIVATION OF CREB AND MAPK IN DORSAL ROOT GANGLIA AND SPINAL CORD

R. R. Ji and C. J. Woolf, NPRG, Dept. of Anesthesia and Cntical Care, MGH and Harvard Medical School, Boston, Massachusetts 02129

Aim of Investigation The cyclic AMP response element binding protein (CREB) and mitogen-activated protein kmase (MAPK) are two critical mediators of activity- and growth factor-induced signal transduction They have been implicated in neuronal plasticity in the hippocampus, such as long term potentiation and memory, after activation by phosphorylation The present study was designed to examine the phosphorylation of CREB and MAPK in the dorsal root ganglia (DRG) and spinal cord after noxious stimulation.

Methods. Using phospho-specific antibodies, activation/ phosphorylation of CREB and MAPK were tested by western blot and im-munohistochemistry in the adult rats or DRG cultures

Results. Under normal condition CREB was present in all DRG neurons, whereas phosphoCREB (pCREB) was only present in some small, TrkA-positive DRG neurons. Intraplantar capsaicm induced a rapid pCREB increase within a few minutes in many small DRG neurons. Electncal stimulation of sciatic nerve at C-fiber strength also increased CREB activation MAPK phosphorylation was transient in DRG after noxious stimulation, followed by a hypophosphorylation In adult DRG cultures both CREB and MAPK were activated by NGF and KCl-mduced depolanzation Moreover, both CREB and MAPK were phosphorylated in dorsal horn neurons after noxious stimulation.

Conclusions. Since CRE sites have been detected in the promotors of many genes, such as BDNF, c-fos, dynorphm, CREB phosphorylation on Ser-133, possibly via MAPK and CaM kmase, may be important for noxious stimulation/inflammation-produced gene expression Acknowledgments Supported by NIH and Roche BioScience

INVOLVEMENT OF CAMP-RESPONSIVE ELEMENT-BINDING PROTEIN (CREB) IN CENTRAL SENS1TIZA-TION FOLLOWING INTRADERMAL CAPSAICIN INJECTION

Jmg Wu. Li Fang*, Qmg Lm, William D. Willis, Dept of Anatomy & Neuroscience, Univ of Texas Medical Branch, Galveston, TX 77550-1069, USA

Aim of Investigation: To investigate the involvement ofcAMP-responsive element-binding protein (CREB) in signal transduction in central changes following mtradermal capsaicm injection. Methods- Sprague-Dawley rats were given an mtradermal injection ofcapsaicm or vehicle in the left paw Animals were sacnficed at 20, 60, 90, and 150 mm after the injection and the lumbosacral segments of the spinal cord were removed The expression of CREB and phosphorylated CREB (p-CREB) was tested by Western blot

Results: The proportion of p-CREB relative to CREB started to increase by 20 minutes and reached a peak level between 20 and 60 minutes following the injection ofcapsaicm The ratio was decreased at 90 mm and back to the control level at 150 minutes. By contrast, the expression of CREB itself did not show an obvious change at any time point after capsaicm injection No significant change was found in either the expression of CREB or p-CREB in animals treated with a vehicle injection at any time point.

Conclusions: CREB is phosphorylated following capsaicm injection. This result suggests that CREB is involved m the mtracellular signal transduction pathways activated following mtradermal cap-saicin injection and that it may play a role in the central sensitiza-tion or longer term changes in gene expression induced by strong peripheral noxious stimulation.

Acknowledgments Supported by NIH grants NS09743 and NS11255.

MULTIPLE SPLICE PATTERNS OF CYCLIC AMP RESPONSE ELEMENT BINDING PROTEIN MESSENGER RNA IN THE RAT NERVOUS SYSTEM

Christian Pietruck. Guo-xi Xie*, Manohar Sharma*, Thomas Meuser, Pamela Pierce Palmer, Dept of Anesthesia, Univ of California, San Francisco, 513 Parnassus Ave, Box 0648, San Francisco, CA, 94143-0648, USA

Aim of Investigation. The cyclic AMP response element binding protein (CREB) is a transcription factor involved in regulating transcription of many important neurotransmitters and regulatory proteins implicated in pain and memory such as c-FOS, synapsm I, dynorphm, and enkephalm The alternative splicing pattern of CREB in different regions of the rat nervous system was to be investigated to gain insight into possibly important tissue specific transcription regulation

Methods: Rat peripheral and central nervous tissue was isolated. CREB mRNA alternative splicing was investigated by an exon-flankmg polymerase chain reaction (PCR) strategy A series of RT-PCR with pnmer pairs flanking all possible alternative splicing sites (corresponding to a genomic region with at least one full exon and two flanking mtrons) was performed Results: PCR has revealed multiple splice patterns in different regions of the rat nervous system. These include some novel transcripts that lack the essential phosphorylation site and a segment of the leucine zipper region, which is crucial for dimenzation and DNA-bmdmg. Some isoforms previously reported as testis-specific were also detected in the rat nervous system. High tissue specificity could be found especially in thalamus, peripheral ganglia, and spinal cord

Conclusions The findings from this study suggest differential splicing patterns of CREB mRNA among different regions of the nervous system Complex expression and functional regulation of CREB in the central and peripheral nervous system, which may influence synaptic plasticity, can be assumed Acknowledgments. Supported by NIH Grant NS56256-01 (Dr Pierce Palmer). Dr Pietruck is funded by the Deutsche For-schungsgememschaft (DFG; Pi3 50/1-1)

EFFECTS OF INTRATHECAL C-FOS ANTISENSE OLIGODEOXYNUCLEOTIDE ON ADJUVANT-INDUCED THERMAL HYPERALGESIA

Shimchi Sugiyo*, Motohide Takemura*, Atsutoshi Tsujio*', Non-fumi Yonehara***, Takashi Nokub** and Yoshio Shigenaga, Depts of "Removable Prosthetics, Pharmacology, and Oral Anatomy, Osaka Univ Faculty of Dentistry, Suita, Osaka 565-0871, Japan

Aim of Investigation To investigate the effects of c-Fos suppression in the spinal dorsal horn on the adjuvant-induced thermal hy-peralgesia by mtrathecalty preadmmistered c-fos antisense oligodeoxynucleotide (ODN)

Method: Male Sprague-Dawley rats (250-300 g body weight) were used (Kean Co. Ltd, Osaka, Japan) Under anesthesia (Nembutal, 50 mg/kg, i p.), a catheter (PE-10 tube) was introduced at around the L4-5 spinal cord through a lammectomy at the L5 vertebra. Antisense or sense ODN complementary to c-fos mRNA was pre-admmistered (25 nM in 20 ul of saline) through the tube Antisense ODN complementary to c-fos with the sequence 5' GAA CAT CAT GGT CGT 3' and sense 5' ACG ACC ATG ATG TTC 3' were phosphoro-thioate analogues (Kurabo Co, Biomedical Branch) Four h later, 200 ul of complete Freund's adjuvand was injected into the plantar surface of the nght hind paw The withdrawal latency to noxious thermal stimulation was examined using Plantar Test (Ugo Basile). In other experiments, effects ofpread-mmistered sense and antisense c-fos ODN on the adjuvant-induced c-Fos expression in the L4-5 spinal dorsal horn were examined using immunohistochemistry

Result Preadmmistered antisense ODN significantly decreased the number of adjuvant-induced c-Fos-immunoreactive neurons in the laminae I/II of the spinal dorsal horn 3 h after adjuvant injection compared to the sense ODN-preadmmistered rat The antisense ODN-preadmmistered rat showed significantly longer withdrawal latency to noxious thermal stimulation in the adjuvant-induced inflamed hind paw than the sense ODN- preadmmistered rat at 3-6 h after adjuvant injection

Conclusion. Intrathecally preadmmistered c-fos antisense ODN suppressed c-Fos expression and attenuated adjuvant-induced thermal hyperalgesia

THE PROMOTER REGION OF HUMAN PREPRO-NOCICEPTIN GENE AND ITS REGULATION BY CYCLIC AMP AND STEROID HORMONES

Guo-xi Xie*, Emi Ito*, Manohar Sharma*, Christian Pietruck, Pamela Pierce Palmer. Dept of Anesthesia, Univ of California, San Francisco, CA 94143, USA

Aim of Investigation. To study the transcnptional regulation of human prepro-nociceptm gene by DNA cloning, sequencing and promoter activity assays

Methods The 5'-flanking region was cloned from adaptor-ligated human genomic DNA libraries by PCR. DNA sequencing and Genebank search were employed to find the binding sites for potential transcription factors Transcription start sites were determined by an "oligo-cappmg" method Promoter activities were measured by luciferase assays

Results The 1 7 kb 5'-flanking fragment of the human prepro-nociceptm gene contains multiple transcription start points and several potential binding sites for transcription factors, including sites for general factors such as TF-IID, AP-2, TCF-1 and C/EBP, and sites for tissue-specific factors such as CREB, ER, and GR Promoter activity assays using luciferase reporter gene vector constructions with the 1 7 kb fragment and a series of deletion mutations demonstrate that the core promoter resides in a region surrounding the major transcription start site - 558, and that the TATA-Box-hke segment displays a weak promoter activity The increase of cellular cyclic AMP levels up-regulates the transcription Glucocorticoid displays negative regulatory effect while es-trogen shows positive effect through their receptor responsive elements ER antagonist or site-mutagenisis ofER and CREB reduce or dimmish ER or CREB enhancer effects, respectively

Conclusions Transcription of human prepro-nociceptm gene is initiated via a non-TATA-promoter and a weak TATA-promoter Factors CREB, GR and ER are involved in the regulation at the transcription level Acknowledgment Supported by NIH Grant NS56256-01

OPIATE TOLERANCE IN MICE IS ASSOCIATED WITH INCREASED EXPRESSION OF VARIOUS TRANSCRIPTION FACTORS

David Clark. Xiangqui Li*, Veterans Affairs Palo Alto Health Care System and Stanford Univ, Palo Alto, CA 94304 USA

Aim of Investigation: To determine if the acquisition of opiate tolerance is associated with evidence of altered gene expression in mouse spinal cord

Methods: Tolerance to the analgesic effects of morphine was accomplished by implanting morphine tablets in mice Protein preparations from the spinal cords of treated and sham operated mice were subjected to Western blot analysis using various transcription factor antibodies Separate tissue was used for immunohistochemistry

Results Morphine pellet implantation caused marked tolerance to the analgesic effects of morphine Western blot analysis revealed that opiate tolerance was associated with a 1 6-fold increase in CREB and 2 1 -fold increase in Phospho-CREB immunorectivity Likewise, levels ofFosB and delta-FosB were Increased 2 2 and 2 0-fold respectively Fos Expression was not significantly changed. Immunohistochemistry revealed that altered levels of transcription factors occurred primarily in the dorsal horns.

Conclusions The dorsal horn of the spinal cord is an important site of action for opiate analgesics In these studies we demonstrate that altered levels of specific transcription factors exist in the spinal cords of opiate tolerant animals These results support the hypothesis that changes in gene expression accompany the acquisition of tolerance to opiates

REGULATION OF tt25 CALCIUM CHANNEL SUBUNIT IN DORSAL ROOT GANGLIA AND SPINAL CORD OF RATS WITH TACTILE ALLODYNIA

ZD Luo. ES Higuera*, *KA Stauderman*, ME Williams*, SR Chaplan Dept of Anesthesiology, Umv of California San Diego, La Jolla, CA 92093, SIBIA Neurosciences, Inc , La Jolla, CA 92037, USA

Aim of Investigation. Sensation may be pathologically altered after peripheral nerve injury such that light touch is intensely painful Spinal administration ofgabapentm selectively reduces this tactile allodynia by an unknown mechanism In vitro, gabapentm binds to the tt25 voltage-dependent calcium channel subunit Thus, nerve injury may result in altered tt25 subumt expression that plays a role in neuropathic pain processing in spinal cord and dorsal root ganglia (DRG) We investigated tt25 subunit expression in spinal cord and DRG in a rat nerve injury model

Methods. Unilateral L5/6 spinal nerve ligation (SNL) in rats, allodynia assessment, RNase protection assays, Western blots using tt25 monoclonal antibody.

Results. Rats developed tactile allodynia four days after SNL At one week, tt25 protein was increased over 10 fold in ipsilateral L5/6 DRG, where a26 mRNA was increased 6-7 fold two days after SNL and remained upregulated for at least one week Spinal cord tt25 mRNA and protein levels were only mildly increased one week after SNL Interestingly, spinal cord a25 subunit was of lower apparent molecular mass than DRG tt25 subunit in denaturing gels

Conclusions. Nerve injury results in a26 calcium channel subunit up-regulation in DRG and spinal cord, preceding allodynia onset Differential tt25 isoform expression or post-translational modification may occur m neuronal tissues Thus, a25 subunit regulation may play a role in the development of allodynia, and gabapentm binding to this subunit may have pharmacologic significance Acknowledgments. Supported in part by NIH grants F32HL09848, NS01769 & a HHMI institutional grant

NMDA RECEPTOR CURRENTS AND MRNA EXPRESSION IN ACUTELY ISOLATED SUPERFICIAL DORSAL HORN NEURONS AFTER A PERIPHERAL NERVE LESION

Urban Karlsson*. Kristma Angeby Moller, Johanna Sjodm*, Lilian Wikstrom*, Jacques Nasstrom, Cellular and Molecular Pharmacology, Astra Pain Discovery Unit Sweden, Astra Pain Control AB, S-141 57 Huddmge, Sweden

Aim of Investigation To study the effects of a peripheral nerve lesion on NMDA receptor mediated whole-cell currents and expression of NMDA receptor subunit mRNAs in single superficial dorsal horn (SDH) neurons

Methods The L5 spinal nerve was lesioned in rats (65-75 g) as a model of neuropathy. The rats were tested for increased mechanical sensitivity (see poster by Angeby Moller) The control rats did not undergo any surgery Seven to 14 days after surgery, slices of the L5 region of the spinal cord were enzyme treated and single SDH neurons were isolated Whole-cell currents were evoked by fast application of glutamate in the presence of 10 m M NBQX and 10 m M glycme The neurons were harvested, cDNA synthesized, a primary multiplex PCR and a secondary nested PCR was run Products were analyzed by agarose gel electrophoresis

Results The nerve lesioned rats showed increased mechanical sensitivity The electrophysiological parameters measured (glutamate EC50, block by Mg2+ and ifenprodil, current density) did not differ between the control and the nerve injury population All subunits assayed for; NR1 (splice variants a and b), NR2A, NR2B, NR2C and NR2D were present in both populations No significant changes were found in the frequency of expression of the subunits between the two populations The most common combination in an individual neuron was NRla/b, 2A, 2B and 2D in both populations

Conclusions. The mechanical hypersensitivity in this model of neuropathy appears not to be dependent on changes in either functionality or subunit composition of NMDA receptors of neurons in the SDH

EXPRESSION OF SODIUM CHANNEL tt-SNS/PN3 AND tt-III MRNAS IN THE TRIGEMINAL GANGLION AFTER PERIPHERAL NERVE INJURY

UlfBongenhielm*'2, Jonas Enksson*, Christopher Nosrat*1, Kaj Fried, Dept ofNeuroscience, Dept of Oral & Max. Surgery, Karolmska Inst, Stockholm, Sweden

Aim of Investigation To analyze alterations in mRNA expression of sodium channels tt-SNS/PN3 and tt-III, within the tngeminal ganglion (TG), following peripheral nerve injury; as recent data suggests possible links to some models of neuropathic pain Methods- At an initial surgical procedure under anaesthesia (chloral hydrate, 350 mg kg ', i p ) 12 adult male Wistar rats had the left inferior alveolar nerve tightly ligated (6/0 silk suture) and cut immediately distal to the ligature Recovery was permitted for periods from 3 to 162 days At the end of the recovery periods the animals were anaesthetized as above and the TGs harvested Using in situ hybridization, we analyzed tt-SNS/PN3 and tt-III mRNA expression patterns and their possible alterations after the expenmental procedure within the mandibular division of the TG

Results All the examined TGs expressed tt-SNS/PN3 mRNA in neurons of the mandibular division A rapid decrease at 3 days post-injury was followed by a normalization after approx 2 weeks A majority of the neurons that expressed tt-SNS/PN3 mRNA were small-medium sized (<1200um2) in normal as well as injured ganglia. tt-III mRNA was not detected in control TGs, but was expressed in neurons after axotomy

Conclusion The results show changes in the pattern ofTG tt-SNS and tt-III mRNA expression after injury, which in some respects differs from the pattern seen at spinal levels These changes may contribute to the abnormal electrical activity associated with orofa-cial dysaesthesia

Acknowledgement Supported in part by The Swedish MRC (Proj no 8654)

METABOTROPIC GLUTAMATE RECEPTORS MRNA EXPRESSION IS REGULATED DURING MONOARTHRI-TIS IN RATS

F L. Neto* 2, J Schadrack2, A Berthele2, W Zieglgansberger2, TR TolandJM Castro-Lopes2 'Inst Hist and Embryol, Fac of Medicine and IBMC, Porto, Portugal, Max-Planck-Inst of Psychiatry, Clin Neuropharm , Munich, Germany

Aim of Investigation. Metabotropic glutamate receptors (mGluRs) have been thought to participate in acute pain and inflammation In the present study, we intended to assess the occurrence of plastic changes in the expression of these receptors, in a rat model of chronic pain

Methods. In situ hybridization with subtype-specific "S-labelled oligonucleotide probes was performed in thalamic coronal sections of controls and monoarthntic (MA) rats at 2, 4 and 14 days (2d, 4d anri 14d"i of the disease The level of mRNA expression of each mGluR was determined ipsi- and contralaterally to the inflamed paw in individual thalamic nuclei, by autoradiography in emulsion-dipped sections. Grain densities (grain area/cell area) in the regions of interest were averaged (N=6 per group), expressed as x fold of background density values and statistically analyzed.

Results: MA rats showed behavioural and physical signs of painful arthritis. The mGluRl mRNA expression exhibited a biphasic timecourse pattern during MA. Changes were higher at 2d with average decreases of 40% of control values in the ventrobasal complex (VB) and 47% in the posterior thalamic (Po) nuclei, contralaterally to the inflamed joint. At 4d the values were in the control range while at 14d they were again reduced. The signal for the mGluR4 mRNA decreased at 4 and 14d ofMA, changes being more pronounced ipsilaterally in the VB (36% and 37%, for the two timepoints, respectively) and Po (33% and 34%). As to mGluR7, the mRNA expression was reduced at 2d in the VB (43%), and at 4 and 14d ofMA in the VB (41% and 35%, respectively) and Po (52% and 34%). In contrast, mGluR3 mRNA showed bilateral increased expression in the reticular thalamic nucleus (Rt) at all timepoints of the disease, with maximum values (101 %) at 2d of disease.

Conclusions: The mRNA expression for the mGluR subtypes studied was differently time-regulated in the thalamus during the development ofMA. The reduced expression of the mGluRs in the VB and Po nuclei, and the increases in the Rt, may counteract the increases of noxious input.

Acknowledgments: Supported by PRAXIS XXI/BD/9795/96 (Portugal) and SFB 391/C9 (Germany).

FOS EXPRESSION IN THE DORSAL COLUMN NUCLEI OF THE RAT FOLLOWING SPINAL CORD INJURY

Danielle Butler2, Kevin Keav2. Philip Siddall2, Dept of Anatomy and Histology, Univ of Sydney, Dept of Anaesthesia and Pain Management, Royal North Shore Hospital, NSW, 2065, Australia

Aim of Investigation: Allodynia is a common problem following spinal cord injury (SCI). It may be due to a change in responsive-ness of neurons at a spinal and/or supraspinal level. We used a contusion model of SCI to determine whether allodynia was associated with a change in neuronal activity in the DCN as measured by Fos immunoreactivity.

Methods: Under halothane anesthesia, rats were subjected to one of the following procedures: laminectomy and contusion injury to the spinal cord at T10; 2. removal of the T10 spinous process; or 3. no surgery. Prior to surgery, vocalisation threshold was determined using graduated Von Frey hairs. Prior to perfusion, half of the animals in each group were stimulated with a Von Frey filament for 1 hour. Immunohistochemical techniques were used to visualise Fos-like immunoreactivity in the DCN.

Results: It was found that: 1. stimulation resulted in a significant increase in DCN Fos expression in all groups; 2. SCI was associated with an increase in basal level of Fos expression in the gracile nucleus when compared to normal animals; 3. SCI and surgery were associated with an increase in evoked Fos expression when compared to normal animals, particularly in the cuneate nucleus; 4. allodynia was not associated with a difference in evoked or basal DCN Fos expression.

Conclusions: SCI results in changes in evoked and basal activity in DCN neurons as measured by Fos immunoreactivity. However the presence of allodynia does not appear to be specifically related to the level or distribution of DCN neuronal activity.

9th WORLD CONGRESS ON PAIN, 1999, Vienna, Austria, p. 4 - 8

   

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