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© 1999 Nature America Inc. • http://neurosci.nature.com©

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18 nature neuroscience • volume 2 no 1 • january 1999

articles

In various species, peripheral injury produces long-lasting sen-sitization of central and peripheral neurons representing theaffected area1,2. In Aplysia, memory-like traces (lasting days orweeks) of noxious peripheral stimulation include enhancement ofcentral synaptic transmission3–5 and long-term hyperexcitabilityof the central soma and peripheral branches of nociceptive sen-sory neurons4–6. In vitro studies of these same neurons, usingserotonin to mimic sensitizing stimulation, have linked long-term synaptic facilitation to synthesis of cAMP and stimulation ofprotein kinase A (PKA)7, whose catalytic subunit translocatesinto the nucleus8. There it activates a CRE binding protein(CREB1a), thus altering gene expression9–11. Studies in rodentsand Drosophila indicate that the cAMP-PKA-CREB pathway isimportant in consolidating long-term memory and inducingtranscription-dependent synaptic potentiation12,13.

Much less attention has been paid to the cGMP-PKG path-way for transcription-dependent plasticity. Indeed, relatively lit-tle is known about the distribution, activity and substrates ofPKG in neural tissue14 or about the role of PKG in regulatinggene transcription in any tissue15. Nevertheless, the cGMP-PKGpathway has been implicated in activity-dependent neural alter-ations lasting hours16,17, and it may trigger some forms of per-sistent pain18. Also, PKG can regulate gene expression in thebrain19–21. In baby hamster kidney cells, PKG is actively import-ed into, but not exported from the nucleus22. There it can regu-late gene expression via several sequence elements15. In contrast,PKA diffuses into the nucleus8,23 and is rapidly expelled byendogenous protein kinase inhibitor24. The active import andsequestration of activated PKG in the nucleus make it an attrac-tive candidate for inducing long-term memory.

Within individual cells known to contribute to memory ofperipheral injury, we tested the hypothesis that the NO-cGMP-PKG pathway is necessary and sufficient to trigger transcription-dependent, nociceptive sensitization. We found that this highlyconserved signaling pathway, but not the cAMP-PKA pathway, is

critical for the induction of long-term alterations in the samenociceptive sensory neurons of Aplysia that have yielded evidencefor memory-like functions of the serotonin-cAMP-PKA pathway.

ResultsWe first asked if injection of cGMP into individual sensory neu-rons (Fig. 1a) is sufficient to induce LTH of the sensory neuronsoma, as reported for cAMP injection25 (M.R.L. & E.T.W., Soc.Neurosci. Abstr. 22, 1445, 1996). Enhanced soma excitability is asso-ciated with an increased probability of soma-generated afterdis-charge triggered by spike trains arriving from the periphery26. Thisafterdischarge can greatly amplify the output of the sensory neu-rons. Our primary measure of excitability was repetitive firing,specifically the number of action potentials evoked by delivery ofa standard one-second depolarizing current pulse into the somathrough the recording electrode (Fig. 1b). Although backgroundexcitability can vary substantially in these cells (Fig. 1c), sensoryneurons tested one day after cGMP injection consistently showedgreater repetitive firing than uninjected cells in the same clusteror cells injected with the cGMP breakdown product, 5´GMP, whichfailed to induce significant LTH (Fig. 1b and c). Separately, weshowed that excitability was not significantly different betweenuninjected cells and cells 24 hours after injection of Fast Green dye(6.2 ± 1.8 spikes; uninjected, 6.8 ± 1.9 spikes; n = 6 sensory clustersand 32 and 36 cells, respectively). Additional preparations showedthat injection of cGMP also caused a long-term decrease in spikethreshold compared to cells injected with Fast Green dye and touninjected cells (cGMP, 1.18 ± 0.05 nA; Fast Green, 1.44 ± 0.07 nA;uninjected, 1.30 ± 0.05 nA; p < 0.05, ANOVA and Newman-Keulstests, n = 85, 42 and 114 cells, respectively, in 13 clusters). Becausethe effect on repetitive firing was more prominent, we subsequentlyfocused on this measure of excitability.

A previous study25 demonstrated long-term depression of out-ward currents following iontophoresis of cAMP into the sensoryneuron soma. We found that pressure injection of either cAMP

Cyclic GMP pathway is criticalfor inducing long-term sensitizationof nociceptive sensory neurons

Matthew R. Lewin and Edgar T. Walters

Department of Integrative Biology, Physiology and Pharmacology, University of Texas-Houston, 6431 Fannin St., Houston, Texas 77030, USA

Correspondence should be addressed to E.T.W. ([email protected])

Noxious stimulation can trigger persistent sensitization of somatosensory systems that involvesmemory-like mechanisms. Here we report that noxious stimulation of the mollusc Aplysiaproduces transcription-dependent, long-term hyperexcitability (LTH) of nociceptive sensoryneurons that requires a nitric oxide (NO)-cyclic GMP-protein kinase G (PKG) pathway. Injectionof cGMP induced LTH, whereas antagonists of the NO-cGMP-PKG pathway prevented pinch-induced LTH. Co-injection of calcium/cAMP-responsive-element (CRE) blocked both pinch-induced LTH and cAMP-induced LTH, but antagonists of protein kinase A (PKA) failed to blockpinch-induced LTH. Thus the NO-cGMP-PKG pathway and at least one other pathway, but notthe cAMP-PKA pathway, are critical for inducing LTH after brief, noxious stimulation.


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nature neuroscience • volume 2 no 1 • january 1999 19

or cGMP produced hyperexcitability 24 hours later (LTH), butthe necessary concentration was substantially less for cGMP thanfor cAMP (Fig. 2a). The threshold concentration of cGMP in themicropipette for inducing LTH (compared to uninjected cells)was ~1 mM, whereas the threshold concentration of cAMP wasover 100 mM. The actual intracellular concentrations after injec-tion were not determined. LTH induced by injection of cGMPwas blocked by co-injection of the specific PKG inhibitor, KT5823(Fig. 2b), suggesting that cGMP’s effect was mediated by PKG.Injection of KT5823 had no significant effect by itself. Co-injectionof KT5823 had the opposite effect on induction of LTH by cAMP(Fig. 2b), indicating that cGMP and cAMP induce LTH via dif-ferent pathways, and suggesting that PKG inhibits the cAMP path-way. Co-injection of a PKA inhibitor, PKI-A, with cGMP causeda significant enhancement of LTH (cGMP, 210 ± 26%; cGMP andPKI-A, 316 ± 26%; p < 0.02, n = 11 and 21 cells, respectively; datanormalized to average excitability of uninjected cells in the sameganglia). Therefore, cGMP does not induce LTH by cross-activa-tion of PKA, and both cGMP and cAMP may inhibit inductionof LTH by the opposite protein kinase.

We next demonstrated that LTH produced by injection ofcGMP depends on changes in protein synthesis and gene tran-scription. The protein synthesis inhibitors anisomycin or eme-tine, or the transcriptional inhibitor actinomycin D, blocked LTHinduced by cGMP injection (Fig. 3a). Because PKG can regulategene expression via CRE (and other response elements) in somecells15, we then tested the possibility that cGMP induces LTH inAplysia sensory neurons via a pathway requiring binding of CREBto CRE. We extended to long-term excitability changes the find-ing that injection of exogenous CRE into these sensory neuronscan block cAMP-dependent long-term synaptic facilitation9. Co-injection of wild-type, but not mutated, CRE effectively blockedLTH induced by cAMP injection (Fig. 3b). However, co-injec-tion of the same concentration of CRE with cGMP did not inter-fere with the induction of LTH by cGMP (Fig. 3b). This suggeststhat cGMP induces LTH by acting on nuclear factors other thanCREB, indicating once again that cGMP and cAMP induce LTHthrough separate pathways.

Because cGMP synthesis is commonly activated by NO, we askedwhether a NO-cGMP-PKG pathway is necessary for LTH of Aplysiasensory neurons induced by natural stimulation. Neurons in eachpleural sensory cluster are primary nociceptors27 that are selective-ly sensitized by noxious stimulation within their peripheral recep-tive fields4,6. To sensitize large numbers of sensory neurons afterselective drug treatment, we used a split-body preparation (Fig. 4a)that permitted the activation and modulation of most cells in eachcluster (Fig. 1a) by brief, extensive pinching and superficial cutsacross the ipsilateral body half, which responded with strong reflexcontractions. The nerves were then cut close to the body wall (whichwas immediately removed), leaving the proximal nerve segmentssufficiently long so that at 24 hours, slow axoplasmic induction sig-nals conveyed by retrograde transport from sites of axotomy wouldnot contribute to any LTH observed28–30. The pinch procedure pro-duced significant LTH of sensory neurons (pinched half, 11.1 ± 2.5spikes; unpinched half, 7.0 ± 1.4 spikes; p < 0.002, t-test, n = 10preparations with 7–17 cells sampled in each cluster).

Application of the NO synthase inhibitor NG-nitro-L-arginine(L-nitro) by unilateral perfusion of central pleural-pedal gangliaand attached body wall through the vasculature produced signif-icant inhibition of LTH compared to that occurring during per-fusion with the inactive isomer NG-nitro-D-arginine (D-nitro;Fig. 4b). Similar inhibition occurred when only the ganglia wereperfused with inhibitor (Fig. 4b), indicating that a required site

of NO production during induction of LTH by peripheral stimu-lation is within the central ganglia. To compare the effects of var-ious inhibitors, we normalized the response of each cell to theaverage response of cells exposed to the effects of the pinchsequence but no other treatment in each preparation. This con-trolled for variation in background excitability (for example,

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Fig. 1. Injection of cyclic GMP induces long-term hyperexcitability(LTH). (a) Pleural-pedal ganglia and enlarged view of pleural sen-sory cluster. Dark cells in cluster illustrate typical pattern followingco-injection of Fast Green and cGMP (or other agents) into individ-ual sensory neurons. (b) Repetitive firing during a 1-second intra-cellular test pulse in sensory neurons injected 24 h earlier withcGMP or 5´GMP. (c) Mean (± standard error) responses from twoanimals exhibiting opposite extremes of background excitability.* p < 0.05 compared to uninjected cells (Un) by ANOVA andDunnett’s tests within the same animal.


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Fig. 1c). Significantly less excitability was found 24 hours aftertreatment in cells treated for only 20 minutes before and 1.5 hoursafter pinch with another nitric oxide synthase inhibitor, NG-methyl-L-arginine (L-NMMA), or with the general guanylylcyclase inhibitor, methylene blue (MeBlue), or with the selectiveinhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3,-a]-quinoxalin-1-one (ODQ), relative to untreated cells in contralat-eral, pinched halves (Fig. 4c). Similar inhibition of LTH wasobserved in cells treated with the highly permeant PKG inhibitor,Rp-8-pCPT-cGMPS. Moreover, significantly less LTH wasobserved in cells injected before pinch with the specific PKGinhibitors KT5823 or cGMP-dependent protein kinase inhibitorypeptide than in either surrounding uninjected cells or nearby cellsinjected with Fast Green dye alone before pinch (Fig. 4c). In con-

trast to the effects of the PKG inhibitors, cells injected with a spe-cific PKA inhibitor peptide (Methods) showed no significant dif-ference in LTH from uninjected cells whose receptive fields werepinched, or from cells injected with Fast Green alone (Fig. 4d).Similar results were obtained with the membrane-permeant PKAinhibitors Rp-8-CPT-cAMPS and H-89 (Fig. 4d). Both injectionof PKI-A and perfusion with Rp-8-CPT-cAMPS had other phys-iological effects on sensory neurons, attenuating immediate hyper-excitability induced by cAMP injection or by serotoninapplication, and reducing the expression of LTH induced by axo-tomy several days earlier31. We have not yet examined other effectsof H-89. These results show that activation of the NO-cGMP-PKG pathway is required for pinch-induced LTH, whereas acti-vation of PKA is not.

Fig. 2. Comparison of LTH induced by injection of cGMP and cAMP. (a) Dose–response relationships (mean ± standard error) for LTH pro-duced by injection of cGMP and injection of cAMP. n, number of cells (from 23 sensory clusters total). The standard error for the uninjectedcells is smaller than the symbol (square on left). (b) Opposite effects of the PKG inhibitor KT5823 on LTH induced by cGMP (30 mM) andby cAMP (400 mM). * p < 0.05, t-test comparing LTH after cyclic nucleotide injections with and without KT5823.

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Fig. 3. LTH induced by cGMP injection requires macromolecular synthesis that is independent of CREB activity. (a) Effects (mean ± standard error)of macromolecular synthesis inhibitors on cGMP-induced LTH. Aniso, anisomycin; Emet, emetine; Actino, actinomycin D. * p < 0.05 compared to allother groups (ANOVA and Newman-Keuls tests). (b) CRE co-injection blocks cAMP-induced but not cGMP-induced LTH. CREwt,wild-type CRE;CREmut, mutated CRE. * p < 0.001, t-test comparing LTH after wild-type and mutated CRE co-injections.

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Because CREB activity in many cell typescan be influenced by various protein kinas-es13,32, including PKG15, we examined theeffects of CRE applied before injection onpinch-induced LTH of sensory neurons. CREinjection blocked the LTH induced by pinch(Fig. 4e). This result, combined with the failureof PKA inhibitors to interfere with the induc-tion of LTH by pinch (Fig. 4d) and the failureof CRE co-injection to block cGMP-inducedLTH (Fig. 3b), suggests that, in addition to aPKG pathway, a second (PKA-independent)pathway contributes to the induction of LTHvia activation of CREB. It remains possible,however, that when activated by pinch, PKGinduces LTH via stimulation of CREB, where-as, when activated artificially by injectedcGMP, PKG induces LTH by a different mech-anism that bypasses CREB.

DiscussionThese data indicate that the cGMP-PKGpathway is critical for inducing long-term,transcription-dependent alterations in neu-rons known to encode a form of long-termmemory, in this case, central memory ofperipheral injury4,6. Both cGMP and PKGhave been implicated in regulation of neu-ronal gene expression19–21. In addition,extracellular application of guanylyl cyclaseinhibitors to brain regions in rats33 andsheep34 blocks long-term memory forma-tion, but the link between cGMP and anydependence of these forms of memory onalterations of macromolecular synthesis wasnot explored. Indeed, the assumption wasthat activation of PKG during memory for-mation enhances synaptic release of gluta-mate34, rather than altering gene expressiondirectly within the same cell. In addition,cGMP and PKG contribute to early long-term synaptic potentiation (LTP) at hippocampal synapses17, and the NO-cGMP-PKG pathway may contribute to late, tran-scription-dependent LTP (Y.-F. Lu and R.D.Hawkins, Soc. Neurosci. Abstr. 24, 1074,1998). Our study provides the first directevidence that long-term, transcription-dependent alterations require PKG activationwithin the same neurons.

Although our findings provide strong evi-dence for a critical role of PKG in the induc-tion of LTH, several questions remain. Oneconcerns the sources of NO and sites of cGMPsynthesis and PKG activation. Effective inhi-bition of LTH when various antagonists wereapplied exclusively to the central nervous system indicates thatrequired NO production and PKG activation occur within thepleural-pedal ganglia. Moreover, blockade by intracellular injec-tion of antagonists indicates that cGMP synthesis and PKG acti-vation occur within the sensory neurons. Although NO inducescGMP elevation in the sensory neurons (B. Armitage, N. Buttnerand S.A. Siegelbaum, Soc. Neurosci. Abstr. 17, 1096, 1991) and

both NOS activity35–37 and cGMP activation of protein kinas-es38 occur in the Aplysia nervous system, it remains to be demon-strated biochemically that a NO-dependent cGMP-PKG pathwayis activated in these cells by noxious stimulation. A potentialproblem for measurements within identified neurons is that thesites of PKG activation may be in the complex, heterogeneousneuropil, rather than in the accessible, identifiable somata.

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Fig. 4. NO-cGMP-PKG pathway and a CREB-dependent pathway, but not PKA, arerequired for induction of LTH by brief noxious stimulation. (a) Split-body preparation.Drugs were applied by arterial perfusion or intracellular pressure injection to one gan-glion, and both body halves were pinched. (b) NG-nitro-L-arginine (L-nitro) inhibits LTHinduction compared to NG-nitro-D-arginine (D-nitro) when applied to both the gangliaand body wall or just to the ganglia. (c) Reduction of LTH (mean ± standard error) byblockers of the NO-cGMP-PKG pathway. (See Methods for full names.) (d) Lack of effectof PKA antagonists on LTH. (e) Reduction of LTH by inhibitors of macromolecular syn-thesis and by CRE injection. All groups were compared with ANOVA and Newman-Keulstests, except for L-nitro versus D-nitro and CREwt versus CREmut, which were comparedwith t-tests. * p < 0.05.

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A second question concerns the interaction of the NO-cGMP-PKG pathway with other pathways during the inductionof LTH. The high concentrations of injected cGMP necessaryfor inducing LTH may be a consequence of the distance betweenthe soma and required sites of PKG activation (as well as rapiddegradation by phosphodiesterases), but high concentrationsmight also be needed to compensate for a lack of activity in par-allel pathways that would be co-activated by natural stimuli. Anecessary contribution from a second pathway was indicated bythe blockade of pinch-induced LTH by CRE injection. The lackof an effect of CRE injection on cGMP-induced LTH and thefailure to block pinch-induced LTH with PKA inhibitors sug-gest that a CREB-dependent pathway that is independent of PKAis involved. The notion that long-term memory inductioninvolves the cooperative interaction of different cellular signal-ing pathways is supported by observations in many preparations.For example, various protein kinases are necessary or sufficientto induce transcription-dependent neural plasticity, includingPKA10,39, MAPK40,41, PKC42 and calcium-calmodulin depen-dent protein kinases32,43. In the nucleus, calcium-dependentprotein kinases may be activated by entry of calcium and/orcalmodulin32,44. These and other potential components (forexample, phosphatases) of additional LTH induction pathwaysare likely to be regulated both by diverse neuromodulatorsreleased during noxious stimulation and by calcium influx dur-ing intense spike activity caused by stimulation of the cell’speripheral receptive field2,4,6.

We were surprised to find that pinch-induced LTH of Aplysiasensory neurons does not depend upon PKA activation, becausethese same cells have been used to demonstrate that a serotonin-cAMP-PKA pathway induces cellular alterations that parallelaversive memory. Noxious stimulation is thought to release sero-tonin onto Aplysia sensory neurons45, serotonin stimulatescAMP synthesis in these sensory neurons8,46, and serotonintreatment can evoke protein-synthesis-dependent LTH in dis-sociated sensory neurons47, so we expected the serotonin-cAMP-PKA pathway to be important for LTH induced by noxiousstimulation. Instead, we found that three different PKAinhibitors failed to impede pinch-induced LTH, even thougheach of three PKG inhibitors blocked induction of LTH. We alsofound that cGMP was more potent than cAMP in inducing LTHwhen injected into individual sensory neurons, and that cGMPand cAMP induce LTH via separate pathways. Furthermore, theenhancement of cAMP-induced LTH by co-injection of a PKGinhibitor and the enhancement of cGMP-induced LTH by co-injection of a PKA inhibitor suggest that these separate LTHinduction pathways are mutually inhibitory.

Although PKA does not seem to be important for inducingLTH in our noxious stimulation protocol, it remains to be deter-mined whether it is important for long-term synaptic facilitationunder these conditions. In addition, PKA might contribute tothe induction or consolidation of LTH during repeated or moreprolonged sensitizing stimulation, which has often been used instudies of long-term plasticity in these cells. Our single noxiousstimulation sequence lasted less than two minutes, and transec-tion of the nerves one minute afterwards (which by itself doesnot activate PKA31) would have interrupted continuing neuralactivity from the periphery that might cause prolonged centralrelease of neuromodulators that activate PKA. Activated PKGand other signals, such as MAPK and calmodulin, that are active-ly imported into the nucleus22,32,40 could have a more immediateimpact on gene expression than PKA, which shows only modest,passive entry into the nucleus8, 23.

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MethodsNOXIOUS STIMULATION. Aplysia californica (80–200 g) were anesthetized,dissected, cannulated, and the anesthetic removed as described4,27, leav-ing each half of the animal (Fig. 4a) without viscera or cerebral ganglia,and innervated solely by the ipsilateral pleural-pedal ganglia (Fig. 1a),which were desheathed or catheterized through arteries supplying theganglia. After tying off collateral vessels, perfusion was confirmed withFast Green dye or tiny air emboli. Following drug application, a briefbut spatially extensive noxious stimulation sequence (mimicking theextensive stimulation observed in attacks on Aplysia by lobster, a nat-ural predator) was delivered to the body surface. The entire sequence,consisting of numerous pinches with forceps followed by scattered shal-low incisions of the skin, lasted less than two min. Approximately onemin later, the pedal nerves were cut and the body wall removed. Theganglia were maintained in supplemented L15 culture medium for 24 hat 15oC (ref. 29).

DRUG APPLICATION. Except where indicated, cGMP was injected at aconcentration of 30 mM in the pipette, and cAMP at 400 mM (bothfrom Sigma) with 0.3% Fast Green dye in either distilled water or0.2–0.5 M potassium acetate. All solutions were buffered to pH 7.6.NG-D- and NG-L-nitro-arginine (D-nitro, L-nitro; 1 mM), NG-methyl-L-arginine (L-NMMA, 1 mM), anisomycin (3.5–7.5 µM), actinomycinD (2.5 µM) and methylene blue (MeBlue, 100 µM) (all from Sigma),1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 25 µM; Tocris),Rp-8-pCPT-cGMPS (Rp-cG, 1 mM; Calbiochem), rp-8-CPT-cAMPS(Rp-cA, 1 mM; Biolog) and H-89 (0.1 µM, Calbiochem) were dissolvedin artificial seawater (pH 7.6) and applied extracellularly. D- and L-nitro, L-NMMA, ODQ, Rp-cG, and Rp-cA were applied 20 min beforepinch and washed out 1.5 h after pinch. Anisomycin and actinomycinD were applied 0.5 h before pinch and washed out 3 h later. All otherdrugs were dissolved in 0.2–0.5 M potassium acetate with Fast Greendye (0.3%), and rapidly pressure injected into individual sensory neu-rons with brief (50–200 ms) pulses at 0.5–4 psi until the cell turnedvisibly green. Injected inhibitors included emetine (100 µM, Sigma),KT5823 (1.5–2 µM, Calbiochem), protein kinase inhibitor peptide(denoted PKI-A or PKI(5-22)amide, rabbit sequence, 0.5–1 mM,Sigma), and cGMP-dependent protein kinase inhibitory peptide (PKI-G: Arg-Lys-Arg-Ala-Arg-Lys-Glu; 200 µM, Peninsula Labs). The effec-tive lifetime of PKI-A as judged by its effects on short-termhyperexcitability was 30–60 min. Mammalian somatostatin cAMPresponsive element (CRE, generously provided by P. Dash) was inject-ed intracellularly (10 µg/ml) (wild-type, CREwt, GGC CTC CTT GGCTGA CGT CAG AGA GAG AGT TCT GCA; mutated, CREmut, GGCCTC CTT GGC CTT AAG TGG AGA GAG AGT TCT GCA). CREinjections were deeper than other injections, to promote delivery intothe nucleus. The interval between the first injection and body-wallinjury was never more than 40 min. 5´GMP, anisomycin, emetine, actin-omycin D, D-nitro, L-nitro, L-NMMA, MeBlue, KT5823, PKI-G, PKI-A and H-89 showed no significant long-term effects on excitability inthe absence of noxious stimulation (5–36 cells tested per drug).

EXCITABILITY TESTS. 24 h after treatment, sensory neuron somata wereimpaled and given standard excitability tests28,29. Spike threshold wasdetermined with an ascending series of 20-ms pulses. Repetitive firingwas evoked with a one-second depolarizing pulse at 2.5× the current forthe 20-ms threshold.

DATA ANALYSIS. Normalization to untreated cells in the same sensorycluster controlled for between-animal variability and allowed poolingof cells across clusters for statistical analysis. The test response of each cellwas expressed as the ratio of the number of spikes evoked in that celldivided by the average number of spikes evoked in all untreated cellssampled in the same cluster. In most split-body preparations, both treat-ed and untreated cells were exposed to the effects of peripheral pinch.Data were analyzed with one-way ANOVA followed by two-tailed New-man-Keuls tests (for all between-group comparisons) or Dunnett’s tests(comparing each group to a single reference group). Comparisons ofonly two groups were made with t-tests. For brevity, significant p val-ues are indicated in the figures at the 0.05 level, although the actual lev-els were often lower.


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AcknowledgementsThis work was supported by grants NS35979 and NS35882 from the National

Institute for Neurological Disorders and Stroke. We thank R.T. Ambron,

P.T. Kelly, X. Liao, and F. Murad for suggestions, and C. Brou and G. Rumbly

for assistance.

RECEIVED 30 JULY; ACCEPTED 17 NOVEMBER 1998

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