Molecular genetic approaches to nociceptor development and function

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Molecular genetic approaches to nociceptor development and function A.N.Akopian, N.C. Abson and J.N. Wood The activationof peripheralnociceptors isthesubjectof intensescrutiny, becauseof itssignificance in pain regulation.Genetic approaches,includinghomology cloning,differencecloning and transgenicmanipulationofmiceareprovidingusefulinsightsintonociceptorfunction.Recentwork suggeststhat transcriptionalregulators(for example,islet-l), which are expressedrelatively selectively insensoryneurones,playa crucialroleindefiningcellularphenotype.Differencecloning hasidentifiedgeneswhichencodeboth Iigand-gated andvoltage-gatedion channelsexpressedby small-diametersensoryneurones.Theroleof inflammatorymediatorssuchasNGF in regulating nociceptorfunctionhasbeenclarifiedin mis-expression and deletionstudies.An understanding of the mechanismsthat regulategene expressionin nociceptorsshould provide new waysto manipulatenociceptorsensitivity, withpotentialsignificance for paintherapy. Trends Neurosci. (1996) 19, 240-246

HESTUDYof the molecularmechanismsinvolvedin nociception (the activation of peripheralneurones T in responseto tissue damage)is an increasinglyactive topic of research. The ability to culture and record electrophysiologicallyfrom mammaliansensoryneurones, combined with an increased knowledgeof the pharmacologyof the receptorsystemsthat either activate these cells or transduceresponsesto them in the spinal cord, has led to insights into the action of peripheral activators (for example, bradykinin, ATP, 5-HT, capsaicin and protons) and hyperalgesicmediators (eicosanoids,interleukins,NGF) (Refs 1–5). These approaches have complemented electrophysiological studies using preparationsof spinal cord attached to peripheralneurones and skin’, as well as behavioral tests on animals7, which have demonstrated the importance of peripheral input in the appropriate induction of pain, as well as in the eventual changes in CNSwiring that might result in aberrantpain sensations when prolongednociceptive input occurss’g. One way to understandhow nociceptors workis to look for genes that are expressedselectivelyby these cells. Such molecules seem likely to play a specialized functional role. In this review,we describehow homology cloning for sensory neurone-specific proteins defined geneticallyin simple multicellularorganisms, and difference cloning of sensory neurone-specific transcriptshas led to the identification of novel receptors, channels, structural proteins and transcription factors that are expressed exclusively by subsets of sensory neurones. The selectivity of expression, comA.N.Akopian, bined with evidencethat some of these moleculesplay N.C.Abson and an important role in nociceptor specification or funcJ.N. Wood are at tion supportsthe view that these molecules not only the Deptof are of interest in understandingthe specializedrole of Anatomyand nociceptors, but might also provide new targets for Developmental the developmentof analgesicdrugs. Biology,Universi~ College,Gower Street,London, UK WCIE 6BT.

240

Conserved mechanisms insensory neurogenesis Sensory neurogenesis in flies and worms is determined by a number of identifiedkey regulators,many TINS Vol. 19, No. 6, 1996

Copyright 01996,

of which are transcription factorslO.The regulatory mechanisms involvedin specifyingdifferent cell fates show some similarities across evolution. The factors that determine cell fate might also play an important role in regulatingfunction in adult differentiatedtissue; for example, NGF, as well as being a survivalfactor for developing sensory neurones, regulates nociceptor sensitivity in adult animals (see below). Such observationsput developmentalstudiesat centre stage for our understanding of molecular mechanisms in biology. The search for vertebrate homologies of neurogenic factors has resulted in the discovery of a number of regulators of neuronal fate, the most remarkableof which (so far) is the mammalian achaetescute homologue (Mash-1) gene which is absolutely required, although not by itself sufficient, for the expression of a sympathetic neuronal phenotype in micell. Arethere similarregulatorsthat specifysensoryneurone cell fate? In Tables 1, 2 and 3 we list some of the knownfactorsthat control peripheralneurogenesis in Drosophila melanogaster and Caenorhabditis elegans, together with identifiedvertebratehomologies. A strikingly interesting group of transcription factors, first identifiedby the Rosenfeldgroup,comprises the POU-domainproteins Brn3a, 3b and 3C (Brn3.0, 3.2 and 3.1, respectively),which are relatively selectively expressed in sensory neurones71. A recent immunocytochemical study providesstrong evidence that Brn3a expressionoccurs in migratingneural crest cells and in dividingneuronal precursorsin trigeminal gangliaand dorsal-rootganglia(DRG),suggestingthat the cells that expressthis transcript are predetermined to become sensory, as opposed to sympathetic, neurones72. The Brn3 proteins are the vertebrate homologies of the uric-86 gene product, necessaryfor the development of mechanoreceptive neurones (amongother cell types)in C. elegans. Uric-86 interacts with a LIM-homeodomaintranscription factor, Mec-3, which contains a cysteine-rich Znz+-chelatingdomain characteristic of this class of proteins73.Interestingly, LIM-domain proteins that are relatively selectively

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TABLE 1.Proneuraigenesas regulatorsof peripheralneurogenesis Vertebrate

Invertebrate

Genes

Comments

Refs

Genes

Comments

Refs

Mash-1 (mouse)

Mash-/ requiredfor development of manyolfactory,entericand autonomicneurones ConvertsprospectiveNC and ectodermalcellsto a CNS fate

I 1,12

Achaete-Scute complex(ASC)

BasicHLH proteinsrequiredfor externalsense-organspecification

17

13,14

Achaete,Scute, lethalof scute, asense(Drosophila)

XASHI XASH3a,3b (Xenopus) CASH-1 (chick) HASH-1 (human) Quox-1 (quail)

Math-l

15 16 Expressedin a subpopulationof NC cellsin earlymigration;later expressedby sensorybut not sympatheticneurones

18

(mouse) BasicHLH proteinexpressedin dorsal 20 CNSduringdevelopment

mab-5 (C.elegms)

Specifiespostemtsryonic fatesof cellsin a posteriorregion

19

Atonal(Ato) (Drosophila)

BasicHLH protein;specifies precursorsof chordotonalorgans anda classof photoreceptors BasicHLH proteinthat acts downstreamof mab5; necessary and in somecasessufficientfor the specificationof the neuroblast cellfate

21

lin-32 (C.elegarrs)

22

snail(mouse) sna-1(zebrafish) Xsna(Xenopus) slug(mouse)

Expressedin presumptive NC cellsand roof-platecells; expressioncontinuesin NC cells duringtheir migration;controlsthe epithelialto mesenchymaltransition of pre-migratoryNC cells

23 24 25 26

snail (sna) (Drosophila)

27 Zn2+-fingerprotein requiredto : initiatemesoderminvagination; repressesgenesresponsiblefor differentiationof the neuroectoderm; later expressedin developingNS, 28 first segmentally,then universally

Id (mouse)

29 HLH protein involvedin neural determinationanddifferentiation; expressedin DRG but not sympathetic or adrenal-medullaneurones

extramacrochaetae

Suppressorof sensilladevelopment; HLH proteinthat forms inactive

(Drosophila)

30

heterodimers with products of the ASC and with Daughterless

Proneural genes endow cells with the potential to become neuronal precursors.Abbreviations:DRG, dorsal-root ganglia;HLH, helix–loop-helix; NC, neura~crest;NS, nervoussystem.

who used photobiotin-labelled RNA from various tissues to hybridizeto cDNAfrom a tissue of interest (see Fig. 1). The cDNA-RNAhybrids can then be removed by completing them with streptavidin and extracting the mixture with phenol. The remaining cDNA should be enriched for tissue-specific transcripts,and by subtractingwith RNAfrom a variety of unrelated tissues this approach, combined with the use of PCR, has allowed a number of new tissuespecific genes to be isolated’c. Differential display, a PCR-basedmethod usingpartiallydegenerateprimers, also allows the identification of differentially expressed transcripts77.However,the instant gratification of a range of interestingbands does not always lead to the lasting satisfaction of defining a physiologically significant transcript. Using the longer route of difference cloning, we have identified selectively expressed, mRNA transcripts from rat DRG which contain a variety of nonneuronal cell types as well as small- and large-diameter sensory neurones. We checked that the clones cloning Difference isolated showed a restricted pattern of expression by Another way to identify novel proteins that might in situ hybridization, and analysed partial clones by play an important role in nociceptor function is to sequence analysis. The approach was validated by subtract commonly expressed mRNAsto produce a the identification of transcripts encoding calcitonin cDNA library enriched in sensory neurone-specific gene-related peptide (CGRP), peripherin, carbonic transcripts. The technology for production of a sub- anhydrase, myelin P. and a number of other trantractive library was developedby Sive and St John75, scripts found predominantly in peripheral ganglia’c.

expressedin neuronalcell typeshavebeen also discovered in vertebrates. A LIM-protein, Islet-1, originally discovered by an analysis of the promoter-binding proteins that regulate insulin-gene transcription, is known to be expressed in sensory (and motor) neurones in early development, and to be necessary for their development74.More recently, a paired-box protein DRG-11, also expressedin DRG neurones but not sympathetic neurones, with homology to the C. elegans uric-4 gene product that determines the synaptic connectivity of motor neurones has been identifiedcz,cs. As DRG-11 is expressedboth in sensory neurones and the dorsal-horn neurones that they innervate, it is possiblethat DRG-11 might play a role in specifying the synaptic connectivity of sensory inputinto the dorsalhorn. Asyet, only sensoryneuronespecific rather than nociceptor-specific transcription factors have been identified. An analysis of the regulatory elements of nociceptor-selective genes (see below) is likely to define such molecules.

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TABLE 2. Neuronalgenesas regulatorsof peripheralneurogenesis Vertebrate Comments

Genes

Transmembraneproteinsinvolvedin Notch-1,2, 3 (rat) MotchA,B cell-fatedecisions (mouse) Notch-1 (mouse) TAN-1(human)

Serrate(mouse) TransmembraneIigandsfor Notch jagged(mouse)

Invertebrate Retk

Genes

Comments

31,32 33

Notch (N) (Drosophila)

34-37

lin-12 (C.elegans)

Thoughtto suppressneuronal differentiationin both Drosophila andvertebrates Affectsmanysetsof non-identical 38 homologu&(cellsof similarlineage historythat expressdifferentfates) only someof whichare neural; seeglp-1

39

Delta (Dl) (Drosophila) Serrate (Drosophila)

Humanhomologiesof E(spl)groucho

41

Negativeregulatorof geneswith E or N boxesin their promoters

42

RNA-bindingproteinimplicatedin paraneoplasticsensoryneuropathy Elr A,B,C,D C and D are expressedonly in brain, (mouse,Xenopus as isNRPI in Xenopus andzebrafish)

44

TLEfamily (human) HES-2 (rat)

HUD (human)

45

DNA-bindingproteinthoughtto be 47 C promoterbindingfactor-1 involvedin differentiationof peripheral (CBFI) (mouse, nervoustissue(identicalto RBP-K, KBF2) human)

Transmembraneproteinswhichact asextracellularIigandsfor Notch

Refs

39

40

Enhancerof split SevenbasicHLH transcription complexIE(spl)] factorsrequiredfor normal (Drosophila) sensoryneurogenesis

43

elav(embryonic Requiredfor correct differentiation 46 lethalabnormal and maintenanceof neurones vision)(Drosophila) RNA-bindingprotein involvedin Musashi (Drosophila) externalsensory-organspecification (RBD motif) Suppressorof Hairless [SU(H)] (Drosophila)

Cytoplasmicor nuclearprotein that bindsto the Notch cytoplasmicdomain

48

Neuronalgenesare requiredduringthe selectionof neuronalprecursorsfrom clustersof cellswith the potentialto becomeneurones.They includethe neurogenicgenes.

Among the partial clones identified in this screen, a PzX-likereceptor transcript was identified. Full-length clones encode a functional ATP-gatedcation channel which has many pharmacologicalcharacnamed PzX~, teristics similar to those displayedby the ATP-gated cation channel expressedby rat sensory neurones in culture78. Evidencethat PzX~heteromultimerizeswith a more generallyexpressedP,, receptorwasobtainedby pharmacological studies of oocyte-expressedcRNAs,and an alternatively polyadenylated PzX~transcript was described79.The 3.8 kb PzX~transcript was found to be present in a subset of small-diameterperipherinpositive sensory neurones, and was undetectable in other tested tissuesincluding sympatheticand enteric neurones. This suggeststhat the 3.8 kb PzX~ transcript is restricted to cell types with a predominantly nociceptive modality, where its expression might be physiologicallysignificant in terms of responding to ATP released from damagedtissue. Such a restricted pattern of expressionsuggeststhat we might be able to find a set of transcriptionalregulatorsthat are responsible for the expressionof the PzX~ transcript, and perhaps a unique repertoire of other nociceptor-associated markers.Promoteranalysisof this gene is thus of more than academic interest, as an identification of known or novel transcription factors in the particular combinations that are specific to this cell phenotype might present new avenues for developinganalgesic drugs. In contrast to our understandingof chemical activation of sensory neurones, relatively little is 242

TINS Vol. 19, No. 6, 1996

known about the molecular mechanisms involved in vertebrate mechanoreception or thermoreception, although the technology to examine mechanically activated channels is highly advanced80.A genetic approachto identifyingthe components of mechanosensory transduction, which is of interest for the understanding of mechanical-induced pain, has recently been reviewed81. An additional transcript found to have a restricted pattern of expression in small-diameter sensory neurones encodes an unusualvoltage-gatedNa+channe182.Small-diameter sensory neurones have long been known to express a tetrodotoxin-insensitive (TTXi) voltage-gatedNa+channel, the expression of which is regulatedby NGF (Ref. 83). The role of the channel is uncertain, but some evidence suggeststhat it playsan important role in the transmission of nociceptive information to the spinal cord84. Thus bradykinin-elicitedrelease of CGRP (a peptide found in nociceptors), as well as the depolarizationof dorsalhom cells elicited through C-fibre activation, was insensitive to peripherallyappliedTTX (Ref. 85). The channel, named SNS, has propertiesthat suggestthat it accounts for the TTXi current defined in smalldiameter sensoryneurones. The pharmacology,distribution of expressionand electrophysiologicalcharacteristics of the channel, when expressed in oocytes, are all reminiscent of the small-diameter sensory neurone Na+channel (Fig. 1). Even micromolar levels of the puffer-fishtoxin, ‘lTX, do not block the channel, and the ion selectivity, kinetics of inactivation

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et al. – Nociceptor development

TABLE 3. Neuronal-specificity genesas regulatorsof peripheralneurogenesis Vertebrate Comments

Genes ClassV Pax gene?

No closehomologueidentified

Invertebrate Refe

.

Genes

Comments

Refs

49

Pox neuro(poxn) Specifiesformationof chemosensory 50 asopposedto mechanosensory (Drosophila) organs;expressedin chemosensory-51 organprecursorcellsandtheir daughters

52

ceh-10 (C.elegans)

Homeodomainproteinexpressed 53 in an interneuronereceivingsignals from a thermosensitiveand perhaps photosensitivesensoryneurone

cut (Drosophila)

Homeodomainprotein necessary andsufficientfor the specification of externalsensory(es) organ precursorcell;lossof cutactivity resultsin conversionof es receptorsto chordotonalorgans

pairedgene (Drosophila)

Homeodomainproteinthat regulates 59 expressionof downstream transcriptionfactorsengrailedand gooseberry

60 Brn3a (Bm3.0) Both 3a and 3b widelyexpressedin sensorybut not sympatheticneurones 61 Brn3b(Bm3.2) 62 Brn3c(Brn3.1) 3Cexpressedin a subsetof sensory (ra~ chick) . neurones

uric-86 (C elegans)

63 POU-domainprotein necessaryfor the generationof touch receptors; 64 interactswith mec-3

/s/-1, /s/-2 (rat, chick)

LIM-homeodomainproteinsexpressed 65 in developingsensoryand motor neurones

mec-3 (C.elegans)

LIM-homeodomainprotein necessary 66 for the specificationof touch receptors

Homeodomain and paired-box protein expressed in sensory and dorsal-horn neurones

67

uric-4 (C.elegans)

68 Homeodomainand Daired-box protein requiredf&- correct synaptic inputto ventral-cordmotorneurones

Expressed in a related pattern to thoughtto Prosperoin Drosophila;

69

Prospero (Drosophila)

70 Nuclear homeodomainprotein expressedin subsetsof developing neurones;neededfor correct neuronalspecification; Drosophila mutanthasdelayedor arrested pioneeringof peripheral motor neurones

Chx10 (mouse)

Expressedin anterior opticvesicle and later in inner-nuclearlayerof mature retina,andother regionsof the nervoussystem

CUT (chicle human) cux (mouse)

Homeodomainproteinwith broad expression Homeodomainproteinthat binds to and regulatesNCAM promoter

Phox2(mouse)

Homeodomainprotein expressedin autonomicbut not sensoryganglia; possiblyinvolvedin expressionof (nor)adrenaline-containing neuronal phenotype;bindsto and regulates NCAM promoter

DRG-I I

(rat)

Prox I (mouse)

55,56

55,58

playa role in earlydevelopmentof murineCNS

54,57

Neuronal-specificity genesare requiredforthegeneration ofneuronal subtypes. Abbreviation: NCAM,neuralcelladhesion molecule.

and channel block by heavy metals are reasonably similar. Interestingly, capsaicin-treated animals or capsaicin-treated cultures of DRG neurones all lose most of the SNStranscript.Becausecapsaicinis known to kill a subset of neurones, most of which are associated with C fibres, this suggeststhat the channel is expressedpredominantlyby nociceptors. If this channel is an important contributor to action-potential propagation in small-diameterneurones, then lowering its expression or blocking its action should result in analgesia,without compromisingnormal responses to non-noxious stimuli. Evidencethat prostaglandins can modulate the current–voltagerelationship of the SNS channel has recently been obtained, suggesting that regulation of this channel might underlie some aspects of hyperalgesia (increased sensitivity to

pain)sG,s7. Apossiblemechanism of short-termchannel modulation wouldbe phosphorylation, and dibutyryl cyclic AMPhas been shown to mimic the actions of prostaglandins,suggestinga role for protein kinase A (PICA).Consistent with this possibility, the major intracellularloop of the SNSNa+channel contains five serines,located within PKAphosphorylation motifs82. Interestingly,studiesof other voltage-gatedNa+channels also found in DRG neurones have shown that PKA-mediatedphosphorylation causes a diminution in peak current rather than a shift in the Z–V plot to more-negativepotentials. Taken together, these observations suggestthat the ‘ITXi Na+channel might be an important regulatorof nociceptor sensitivity,even if it does not play a predominant role in action-potential propagation. 17NSVol. 19, NCJ.6,1996

243

DRG

Liver

i poly (A)+ RNA

Kidney

Cerebellum

“-XX ’’-” poly (A)+ RNA

4 SSCDNA

Cortex

“’-’%X’’” poly (A)+ RNA

\ Photobiotinylation

I

I

t Quiagen chromatography Hybridization in solution Separation of SSCDNAfrom RNA–DNA duplexes ~~

Photobiotinylation

Hybridization in solution

1 Separation of SSCDNAfrom RNA–DNA duplexes

/’-’”

Probe filter with DRG cDNA

G-tailing
Probe filter with cortex /

i

/“

and cerebellum .DNA

//

cDNA library construction Lambda zap II I

//

//

+ Replica plating

~ Probe filter with subtracted DRG cDNA

Fig. 1. Procedure to generate a subtractive library. Differentia/screeningwith probes derived from the subtracted library and irrelevant tissue, as well as from dorsal-root ganglia (DRG) allows a battery of DRG-specific transcripts to be identified. The crucial step in this process involves representative amplification of the subtracted transcripts by PCR. Abbreviation: SSCDNA,single-stranded complementary DNA. Adapted from Ref. 76.

In Fig. 2, some of the chemical mediatorsand activators of nociceptors are shown in schematic fashion. Although there is good evidencefor the expressionof B, bradykininreceptors,5-HT3serotonin receptorsand a host of other receptors for prostanoids and other mediators involved in pain induction, as yet only the PZX:J ligand-gatedreceptoris selectivelyassociatedwith a subset of sensoryneurones. Similarly, the lTXi Na+ channel, SNS,implicated as a charge carrierin actionpotential propagation in nociceptors is highly selectively expressed. Given the likely functional significance of these two channels, an analysis of the regulatorymechanisms involvedin their expressionis of considerable interest. Although classical pharmacology has concentrated on receptor–ligandinteractions, many useful drugsactually block expression at the level of transcription. Defining the common regulatoryelements in PZX3 and SNSchannels should help us to identify components of the regulatorysystem that specifynociceptor-selectivetranscriptexpression. Rational screens can then be constructed, using human reporter constructs to find drug candidates that selectivelyblock expression. Transgenic analysis of nociceptor function

The construction and analysis of mouse null mutants is a direct approachto understandingthe role 244

TINS Vol. 19, No. 6, 1996

of putative regulatorsof vertebrate sensory neurogenesis and function. An analysis of null mutants for potential key regulatorssuch as the Brn3 genes should provide evidence of any role for the murine homoIogues of Uric-86 in sensory-neuronespecification. A recent development in the field of transgenic mouse null-mutant technology has been the production of inducible knockouts, where the bacterial recombinase Cre is used to excise sequencesenclosed within recognized DNAsequencesknown as 10xPsites. This process is analogous to the widely used Flp-recombinasesystem used in Drosophila, which has allowed rapid advances in mutant analysis in this organism to be made. By driving the Cre recombinase with an inducible promoter, such as that for the interferoninduced gene Mxl, inducible knockout animals have been constructed91.This advancein an alreadypowerful technology enables the function of the expressed gene to be analysed in adult mice, without compromising the developmentalmechanisms that might be temporarilydependenton the targetedgene. By combining this expression with tissue-specific inducible Cre-recombinase activity, an even more focused analysis should soon be possible. A related approach to the production of inducible deletions has been developed using the tetracycline promoter to ablate the expression of Caz+–calmodulinkinase type II in adult micegz. Another area in which transgenic manipulation has givenprofoundinsightsinto nociception and pain concernsthe role of hyperalgesicmediators– inflammatory molecules that alter pain thresholds. One important mediator of inflammatory hyperalgesiaappearsto be NGF. Manipulation of NGF levels, mis-expression of NGF,or deletion of its high- and low-affinityreceptors haveclear-cuteffectson painthresholds{. Thus,driving NGF production under the control of a keratin promoter causesalterations in pain thresholds which are particularlyapparentwith noxious mechanical stimulation. In confirmation of these studies, the injection of neutralizing ‘receptorbodies’, where high-affinity receptors are coupled to Fc fragments to neutralize circulating NGF, causes a decrease in noxious pain thresholds in mice; this indicates a tonic role for NGF in defining pain thresholdsg~.NGF is not the only neurotrophin associated with nociception. Brainderivedneurotrophicfactor (BDNF)null-mutantviable heterozygotesshow diminishednoxious thermal pain thresholds.Neurotrophin-4(NT-4)null mutants, however,shownormal pain thresholdsxy.The contribution of differentneurotrophic factors and their receptorsto the survivalof differentsubsetsof peripheralneurones has been recently reviewed’. Overexpression of tumour necrosis factor (TNF), another hyperalgesicmediator, leads to increasedlevels of NGF (Ref. 95), and loweredpain thresholds. In addition, high levels of NGFcan cause abnormal sympathetic interactions with sensory neurones that might contribute to sympathetically induced paingc. Aswith the expression of the channels and receptors involved in nociceptor activation, the modulation of NGF activity on sensory neurones becomes an attractive target for analgesic drug development, particularly as NGFhas a fairly restrictedrange of peripheraltissue targets. Given the timecourse of NGF action, it is likely that both rapid events, probably involving channel modification by mechanisms such as

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et al. – Nociceptor development

2040mv

SubtractedITXi current Currentin the presence of

1

Chemical activators

100 mM TTX

Total current

*

/

W3

5-HT

Ascending

ATP

pathways w

#

CGRP etc.

SNS

#’rm/ /

(TTXi, VGSC)”

NGF PGs f TNF

neuropeptides W/\

- ------

/ ~ A fibres

(VGSC)

Sensitizing signals

-40

II

o

Bz

o

w

DRG-11 /s/-1, 2

!

F’2XI-P2X6 ‘-”T~

SubstanceP

i

Brn3a,b

bradykinin

‘J y

4

~

w “)

-20

,,,

‘

Motor neurones

DRG

10-9 Z.

2

Mv

Spinal cord

SubtractedTTXicurrent Currentin the presence / of 100 MMTrx

3 w

to al current

4

Fig. 2. Schematicrepresentation of noxious input into the spinal cord. Somesmall-diameterunmyelirmtedC fibresare activatedby chemical mediotors that reflect the occurrence of tissue damage (for example, ATP,5-HL bradykinin). Many of the receptors involved in this processhave been defined at the molecular level. The consequences ofreceptor activation depend upon signals that regulate the sensitivityof the cell to actionpotential propagcrtion.Some signals probably involvekinase activation-%w Thjsmight a/ter the excitabilityof either voltage-gated Na+or K+channek to enhance activation by threshold stimuli. The different susceptibilityof fVa+currents to TTXfor small- and large-diameter Na+currents is shown in the boxesgo.Noxiousinput into the spinal cord occurs in superficialIaminae through the release of glutamate and neuropeptides. Known transcription factors, the expression of which correlates with a nociceptor phenotype, are generally also found in myelinated A fibres(DRG-11, 1s1-1,1s1-2, Oct-2, Brn3a),although transcriptionfactors that are differentiallyexpressed in the two populations are likelyto exist(comparedwith Brn3c found only in A fibre-associated cell bodies). Abbreviations: CGRP,calcitonin gene-related peptide; DRG, dorsal-root ganglia; PCS, prostag/andins; TNF,tumour necrosisfactor; TTX~tetrodotoxin-insensitive;VGSC,vokage-gatedNa+channel.

phosphorylation,aswellas changesin geneexpression, play a role in hyperalgesia.It is well documentedthat NGF upregulates the ion flux carried by the TTXi Na+channels on sensory neurones, consistent with a role for this channel in determiningactivation thresholds, as well as the transmission of nociceptive informationg7. In summary, a number of key regulatorsof vertebrate sensoryneurogenesisand function have already been identified by genetic studies, and more will follow. With the increasing subtlety of inducible and tissue-specificknockout technology, the roles of these factors should soon become clear. Asthe regulationof expression of nociceptor-specific channels, receptors and inflammatory mediators becomes better understood, drug developmenttargets that focus on modulation of gene expression,rather than classicalligandreceptor blockade will become an attractive approach to pain therapy. Subtle inhibition of either sensoryneurone activation, aberrant sensory–sympathetic interactions or noxious input into the spinal cord by manipulatinglevelsof gene expressionis the eventual aim of this approach. Selectedreferences

Burgess, G.M.et al. (1989) J. Neurosci. 9, 3314-3325 Bean,B.P. andFriel,D.D. (1990) Ion Channels 2, 169-203 A., Kayser,V. and Guilbaud,G. (1989)Pain 36, 3 Eschalier, 1

2

249-255

Dray,A.andDickenson, A.(1994)in Capsaicin in the Studyof J.N.,cd.),pp.239–255,Academic Press 5 Lewin,G.R.andMendell,L.M.(1993)Trends Neurosci. 16,

4

Pain (Wood, 353-359

Otsuka,M.andYanagisawa, M.(1988)J. Physiol. 395,255-270 7 Ahlgren,S.C., White, D.M. and Levine,J.D. (1992)

6

J. Neurophysiol.68, 2077-2085

8

Woolf,C.J.,Shortland, P.andCoggeshall, R.E.(1992)Nature

9

Woolf,C.J.(1993)Acta Neurochir.SuppL Wien58, 125-130

355, 75-78

10 Le Dourain, N.M., Cacheim, C. and Teillet, M.A. (1992) in SensoryNeurons(Scott, S., cd.), pp.143-171, Oxford University Press 11 Guillemot, F. and Joyner, A.L. (1993) Mech. Dev. 42, 171-185 12 Lo, L. et al. (1994) Perspect.Dev. Neurobio2. 2, 191-201 13 Ferreiro, B. et aL (1992) Mech. Dev. 40, 25-36 14 Turner, D.L. and Weintraub, H. (1994) Genes Dev. 8, 1434-1447 15 Jasoni, C.L. et al. (1994) Development 120, 769-783 16 Ball, D.W. et al. (1993) Proc.Natl Acad. Sci. USA90, 5648-5652 17 Campos-Ortega,J.A. (1993) J. NeurobioL 24, 1305-1327 18 Xue, Z.G., Xue, X.J. and Le Douarin, N.M. (1993) Mech. Dev. 43, 149-158 19 Salser,S.J. and Kenyon, C. (1992) Nature 355, 255-258 20 Shimizu, C. et aL (1995) Eur. J. Biochem. 229, 239-248 21 Jarman, A.P. et aL (1993) Cell73, 1307-1321 22 Zhao, C. and Emmons, S.W_.(1995) Nature373, 74-78 23 Nieto, M.A. et aL (1992) Development 116, 227-237 24 Hammerschmidt, M. and Niisslein-Volhard, C. (1993) Development 119, 1107-1118 25 Mayor, R. et aL (1993) Development 119, 661-671 26 Angela-Nieto,M. et aL (1994) Science 264, 835-839 27 Ip, Y.T., Maggert, K. and Levine, M. (1994) EkfBOJ. 13, 5826-5834 28 Ip, Y.T., Levine,M. and Bier,E. (1994)Development120, 199-207 29 Duncan, M. et aL (1992) Dev. BioL 154, 1-10

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Akopian

et al. - Nociceptor development

30 Cabrera, C.V., Alonso, M.C. and Huikeshoven, H. (1994) Development120, 3595-3603 31 Weinmaster, G., Roberts, V.J. and Lemke, G. (1991) Development113, 199-205 32 Weinmaster, G., Roberts, V.J. and Lemke, G. (1992) Development116, 931–941 33 Lardelli,M. and Lendahl, U. (1993) Exp. CeZZRes.204, 364-372 34 Lardelli, M., Dahlstrand,J. and Lendahl, U. (1994) Mech.Dev. 46, 123–136 35 Franco del Amo, F. et al. (1993) Genomics15, 259-264 36 Ellisen, L.W. et al. (1991) Cell66, 649-661 37 Artavanis-Tsakonas,S., Matsuno, K. and Fortini, M.E. (1995) Science268, 225-232 38 Yochem, J., Weston, K. and Greenwald, I. (1988) Nature 335, 547-550 39 Lindsell, C.E. et al. (1995) Cell80, 909-917 40 Rebay, I. et al. (1991) Cell67, 687-699 41 Stifani, S. et aL (1992) Nat. Genet. 2, 119–127 42 Ishibashi,M. et al. (1993) Eur. J. Biochem.215, 645-652 43 Delidakis, C. and Artavanis-Tsakonas, S. (1992) Proc. Nat/ Acad. Sci. USA89, 8731-8735 44 Marrusich, M.F. et al. (1994) J. Neurobiol.25, 143-155 45 Good, P.J. (1995) Proc.Natl Acad. Sci. USA92, 4557-4561 46 Nakamura, M. et al. (1994) Neuron 13, 67–81 47 Brou, C. et aL (1994) Genes Dev. 8, 2491-503 48 Fortini, M.E. and Artavanis-Tsakonas, S. (1994) Cell 79, 273-282 49 Nell, M. (1993) Curr.Opin. Genet. Dev. 3, 595-605 50 Dambly-Chaudiere,C. et aL (1992) Cell69, 159-172 51 Nottebohm, E. et aL (1994) Neuron 12, 25-34 52 Liu, 1.S.et al. (1994) Neuron 13, 377-393 53 Svendsen, P.C. and McGhee, J.D. (1995) Development 121, Acknowledgements 1253-1262 We acknowledge 54 Blochlinger, K., Jan, L.Y. and Jan, Y.N. (1993) Development generoussupport 117, 441–450 /lore the Wellcome 55 Valarche, I. et aL (1993) Development119, 881-896 Trust(ANAand 56 Blochlinger, K., Jan, L.Y. and Jan, Y.N. (1991) Genes Dev. 5, 1124–1135

JNW) and the 57 Merritt, D.J., Hawken, A. and Whitington, P.M. (1993) Neuron MedicalResearch 10, 741-752 Council(NA),and 58 Tisser-Seta,J.P. et aL (1993) C.R.Acad. Sci. III 316, 1305-1315 helpfidcomments 59 Cai, J. et al. (1994) Mech. Dev. 47, 139-150 60 Xiang, M. et al. (1995) J. Neurosci. 15, 4762-4785 from ourcolleagues 61 Turner, E.E., Jenne, K.J. and Rosenfeld, M.G. (1994) Neuron 12, 205–218 at UCL.

Ninkina, N.N. et aL (1993) NucleicAcidsRes.21, 3175–3182 Finney, M. and Ruvkun, G. (1990) Cell63, 895-905 Xue, D. et al. (1993) Science 261, 1324-1328 Wang, M. and Drucker, D.J. (1995) J. BioZ. Chem. 270, 12646-12652 66 Way, J.C. and Chalfie, M. (1988) Cell54, 5-16 67 Site, T. et al. (1995) Mol. Cell. Neurosci. 6, 280-292 68 White, J. G., Southgate, E. and Thomson, J.N. (1992) Nature 55, 838-841 69 Oliver, G. et al. (1993) Mech.Dev. 44, 3-16 70 Doe, C.Q. et al. (1991) Cell 65, 451-464 71 He, X. et aL (1989) Nature 340, 35-41 72 Fedtsova, N.G. and Turner, E.E. (1995) Mech. Dev. 53, 291–304 73 Way, J.C. and Chalfie, M. (1989) GenesDev. 3, 1823-1833 74 Pfaff, S.L. et aL (1996) Cell84, 309-321 75 Sive, H.L. and StJohn, T. (1988) NucleicAcidsRes. 16, 10937 76 Akopian, A.N. and Wood, J.N. (1995) J. BioL Chem. 270, 21264-21270 77 Liang, P. and Pardee, A.B. (1992) Science14, 967–971 78 Chih-Cheng, C. et al. (1995) Nature 377, 428432 79 Lewis, C. et al. (1995) Nature377, 433–436 80 Hamill, O.P. and McBride, D.W., Jr (1994) TrendsNeurosci.17, 439-443 81 Keman, M. and Zuker, C. (1995) Curr. Opin. Neurobiol.5, 443-448 82 Akopian, A.N., Sivilotti, L. and Wood, J.N. (1996) Nature 379, 257–262 83 Cohen, S.A. and Barchi, R.L. (1993) Int. Rev. Cytol. 137c, 55-103 84 Jeftinija, S. (1994) BrainRes. 639, 125–134 85 Jeftinija, S. (1994) Brain Res. 665, 69–76 86 Gold, M.S. et aZ.(1996) Proc.NatZAcad. Sci. USA93, 1108–1112 87 England, S. et aZ.J. PhysioL(in press) 88 McMahon, S.B. et al. (1995) Nat. Med.8, 774-780 89 Siuciak,J.A. et aL (1995) Soc.Neurosci.Abstr. 21, 1055 90 Kostyuk, P.G., Veselovsky,N.S. and Tsyndreko, A.Y. (1981) Neuroscience 6, 2423-2430 91 Kuhn, R. et al. (1995) Science269, 1427-1429 92 Mayford, M. et al. (1995) Soc.Neurosci.Abstr.21, 1099 93 Davis, B.M. et al. (1993) Neuroscience56, 789-792 94 McMahon, S. and Priestley,J. (1995) Curr. Opin. NeurobioZ.5, 616-624 95 Aloe, L. et al. (1993) GrowthFactors 9, 149-155 96 Davis, B.M. et al. (1994) J. Comp. Neurol. 349, 464-474 97 Aguayo, L.G. and White, G. (1992) Brain Res. 570, 61-67 62 63 64 65

Brain chimeras in birds: application to the study of a genetic form of reflex epilepsy

CesiraBatini is at the Laboratoirede Physiolo&”e de la Moti’cit6,CNRS, Universit61?erre-etMarieCurie,CHU Piti&Safp&r@re, 91 boulevardde Z’H6pital, F75634 A strain of chicken,calledhere FEpi (for Fayoumiepileptic),bearingan autosomalrecessive ParisCedex,France. and Marie-Aim6eTeillet mutation,exhibitsa form of reflexepilepsywith EEG interictalparoxysmalmanifestations and NicoleM. Le generalizedseizuresin responseto eitherlightor soundstimulations. By usingthe brainchimera Douarinare at the Institut technology,we demonstrate here that the epilepticphenotype can be partially or totally d’Embryologic transferredfrom an FEpito a normalchickbygrafiingspecific regionsofthe embryonicbrain.The Cehdaire et containsthe generatorof allepilepticmanifestations whetherthey involvevisual Mol&ulairedu mesencephalon CNRSet du Col12ge or auditory neuronalcircuits,with the exceptionof the abnormal EEG which is transmitted de France,49 bis exclusivelyby telencephalicgrafts.This analysissupportsthe hypothesisthat certainforms of avenuede [a Belle Gabrielle,F94736 humanand mammalianepilepsies havea brainstemorigin. Nogent-sur-Mame Trends Neurosci. (1996) 19, 246-252 Cedex,France. Robe?tNaquet is at Originally, this method involves heterospecific RAINCHIMERASare constructed by exchanging the InstitutA1/ted definite regions of the neuroepithelium between associations of tissues between two species of birds, FessardCNRS, F91198 Gi~sur- two avian embryos of either the same or different the quail and the chick, and usesthe unique structure YvetteCedex, species at developmental stages preceding the vas- of the quail interphase nucleus as a marker to follow cell migrations duringembryogenesis14.The fact that France. cularizationof the encephalic vesiclesl.

Cesira Batini, Marie-Aim6eTeillet,Robert Naquet and Nicole M. Le Douarin

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