Reduced Acetylcholinesterase (AChE)

13 Pages·1997·2.03 MB·English

TheJournalofNeuroscience,February1,1997,17(3):891–903
Reduced Acetylcholinesterase (AChE) Activity in Adrenal Medulla
and Loss of Sympathetic Preganglionic Neurons in TrkA-Deficient,
But Not TrkB-Deficient, Mice
Andreas Schober,1 Liliana Minichiello,2 Markus Keller,3 Katrin Huber,1 Paul G. Layer,3 Jose´ L. Roig-Lo´pez,4
Jose´ E. Garcı´a-Arrara´s,4 Ru¨diger Klein,2 and Klaus Unsicker1
1DepartmentofAnatomyandCellBiologyIII,UniversityofHeidelberg,D-69120Heidelberg,Germany,2European
MolecularBiologyLaboratory,DifferentiationProgramme,D-69117Heidelberg,Germany,3DepartmentofZoology,
TechnicalUniversityofDarmstadt,D-64287Darmstadt,Germany,and4DepartmentofBiology,UniversityofPuertoRico,
RioPiedras,PuertoRico000931-3360
TrkA high-affinity receptors are essential for the normal devel- cord ((cid:50)40%). Other markers of the adrenal medulla and its
opmentofsympatheticparavertebralneuronsandsubpopula- innervation, including various neuropeptides, chromogranin B,
tions of sensory neurons. Paravertebral sympathetic neurons secretogranin II, amine transporters, the catecholamine-
and chromaffin cells of the adrenal medulla share an ontoge- synthesizingenzymestyrosinehydroxylaseandPNMT,synap-
netic origin, responsiveness to NGF, and expression of TrkA. tophysin, and L1, essentially were unchanged. Interestingly,
Whichaspectsofdevelopmentoftheadrenalmedullamightbe AChEimmunoreactivityappearedunaltered,too.Preganglionic
regulatedviaTrkAareunknown.Inthepresentstudywedem- sympatheticneurons,incontrasttoadrenalmedullarycells,do
onstrate that mice deficient for TrkA, but not the neurotrophin not express TrkA. They must, therefore, be affected indirectly
receptorTrkB,showanearlypostnatalprogressivereductionof by the TrkA knock-out, possibly via a retrograde signal from
acetylcholinesterase (AChE) enzymatic activity in the adrenal chromaffincells.OurresultssuggestthatsignalingviaTrkA,but
medulla and in preganglionic sympathetic neurons within the notTrkB,maybeinvolvedinthepostnatalregulationofAChE
thoracic spinal cord, which are also significantly reduced in activityintheadrenalmedullaanditspreganglionicnerves.
number. Quantitative determinations of specific AChE activity Key words: adrenal gland; spinal cord neurons; acetylcho-
revealedamassivedecrease((cid:50)62%)intheadrenalglandand linesterase;chromaffincells;neurotrophinreceptors;knock-out
a lesser, but still pronounced, reduction in the thoracic spinal mice
TrkAisahigh-affinityneurotrophinreceptorthatmediatesessen- and Levi-Montalcini, 1979; Mu¨ller and Unsicker, 1986), and ex-
tial actions of nerve growth factor (NGF) on subsets of sympa- pression of TrkA (Snider, 1994; this study). It is not clear, how-
thetic and sensory neurons as well as CNS forebrain cholinergic ever,whichaspectsofdevelopmentoftheadrenalmedullamaybe
neurons (Thoenen and Barde, 1980; Levi-Montalcini, 1987; regulated via TrkA. Transgenic mice deficient for each of the
Barde,1989;Snider,1994).Inrodents,targetedmutationsofthe knownmembersoftheTrkreceptorfamilyhavebeengenerated
genescodingforTrkA(Smeyneetal.,1994),NGF(Crowleyetal., recently(Kleinetal.,1993,1994;Smeyneetal.,1994)(forreview,
1994)orblockadeofendogenousNGFwithneutralizingantibod- see Klein, 1994; Snider, 1994). Whether the adrenal medulla is
ies (Angeletti et al., 1972; Thoenen, 1972) cause the loss of affectedinanyoftheseknock-outshasnotbeenreported.
paravertebral sympathetic neurons, thereby demonstrating the Beyondcellsurvival,transmittersynthesis,andneuritegrowth,
essentialrolesofTrkAandNGFintheirdevelopment. TrkA-mediated mechanisms have long been known to regulate
Paravertebral sympathetic neurons and the neuroendocrine expressionofcellsurfacemoleculesas,e.g.,neuralcelladhesion
chromaffincellsoftheadrenalmedullaandparagangliasharean molecule (N-CAM) (Prentice et al., 1987), NGF-inducible large
origin from the neural crest (cf. Unsicker, 1993), responsiveness external protein (NILE) (McGuire et al., 1978), and various
toexogenousNGFinvitroandinvivo(Unsickeretal.,1978;Aloe proteases (Machida et al., 1989, 1991). Acetylcholinesterase
(AChE; E.C. 3.1.1.8) is a prominent intra- and extracellularly
locatedenzymeincholinergicandmanynoncholinergicneurons,
ReceivedSept.9,1996;revisedNov.1,1996;acceptedNov.11,1996.
including chromaffin cells (Lewis and Shute, 1969; Massouli´e et
ThisstudywassupportedbyDeutscheForschungsgemeinschaft(SFB317/C8/D4,
SFB269/A2).L.M.issupportedbyalong-termEuropeanMolecularBiologyfellow- al., 1993). Its principal function in cholinergic tissues is the hy-
ship. J.E.G.A. and J.L.R.L. were funded by the Minority International Research drolysisofacetylcholine(Massouli´eetal.,1993),butotherfunc-
TrainingprogramofNationalInstitutesofHealth–FogartyInternationalCenterand
tionsalsohavebeendiscussed(Small,1989;LayerandWillbold,
National Institutes of Health Grant RR-8102-18. We thank Richard Hertel and
Martin Barth for their expert technical assistance. We thank Drs. N. Wolf, K. 1995). NGF has long been known to regulate in vitro acetylcho-
Krieglstein,N.Kahane,andC.Kalcheimforsharingunpublishedresultsonadrenal linesteraseactivityinPC12pheochromocytomacellsandadrenal
TrkB expression. Antibodies to AChE, L1, chromogranin B, secretogranin II,
chromaffincells(forreferences,seeDiscussion).
VMAT-1,andVMAT-2weregenerouslyprovidedbyDrs.J.Massoulie´,A.Faissner,
W.Huttner,andM.Hannah. Adrenalmedullarychromaffincellsreceiveaprominentcholin-
Correspondence should be addressed to Dr. Klaus Unsicker, Department of ergicinnervationfrompreganglionicsympatheticneurons,which,
AnatomyandCellBiologyIII,UniversityofHeidelberg,ImNeuenheimerFeld307,
D-69120Heidelberg,Germany. intheirvastmajority,arelocatedintheintermediolateral(IML)
Copyright(cid:113)1997SocietyforNeuroscience 0270-6474/97/170891-13$05.00/0 columnofthethoracicspinalcord(Kesseetal.,1988;Stracketal.,
892 J.Neurosci.,February1,1997,17(3):891–903 Schoberetal.•TrkA-MediatedRegulationofAChEActivity
1988; Pyner and Coote, 1994a,b) (for review, see Parker, 1996). Table1.Polyclonalprimaryantibodiesusedforimmunocytochemistry
Nervefibers,presumablyofpreganglionicorigin,canbefoundin
theratadrenalglandasearlyasembryonicday(E)15(Millarand Antibody Dilution Reference
Unsicker, 1981). These nerve fibers become AChE-positive dur-
NeuropeptideY 1:500 Garc´ıa-Arrar´asetal.,1992
ing the first postnatal week (Millar and Unsicker, 1981), and a
Somatostatin 1:3000 Garc´ıa-Arrar´asetal.,1984
functional innervation of chromaffin cells, i.e., discharge of cat-
Met-enkephalin 1:500 Dr.Barreto-Estrada,unpublisheddata
echolamines by neurogenic stimuli, commences in rat at the end
VIP 1:1000 Garc´ıa-Arrar´asetal.,1987
ofthefirstweek(Slotkin,1986;Parkeretal.,1988).
Galanin 1:1000 D´ıaz-Mirandaetal.,1996
The present study shows that mice deficient in the TrkA, but
Synaptophysin 1:100 Sigma-AldrichGmbH,Germany
not the TrkB receptor, display a significant reduction in AChE
AChE 1:1000 Marshetal.,1984
activityintheadrenalmedullaanditspreganglionicinnervation.
L1 1:1000 Faissneretal.,1985
Inaddition,numbersofpreganglionicsympatheticneuronsinthe
TH 1:500 EugeneTech,OR
IMLcolumnofthespinalcordarereducedsignificantlyinTrkA-
PNMT 1:1000 Incstar,Stillwater,OK
deficient,butnotTrkB-deficient,animals.
ChromograninB 1:500 Rosaetal.,1985
SecretograninII 1:200 Rosaetal.,1985
MATERIALS AND METHODS
VMAT-1 1:100 Dr.Hannah,unpublisheddata
TrkA-andTrkB-deficient((cid:50)/(cid:50))andwild-type((cid:49)/(cid:49))miceaged0–12d VMAT-2 1:2000 Dr.Hannah,unpublisheddata
wereused.Numbersofanimalsperphenotypewerepostnatal(P)P0,n(cid:53)
15; P3, n (cid:53) 15; P7, n (cid:53) 25; and P12, n (cid:53) 25. The TrkA and TrkB
knock-out mice were generated by breeding heterozygous mutant mice
kept on a mixed 129/sv (cid:51) C57BL/6 background. Standard procedures of0.12Mcoppersulfate,3.0mlof0.16Mpotassiumferricyanide,and0.6
(Lairdetal.,1991)wereusedforthegenomicDNAextractionfromtail ml of 10(cid:50)3 M tetraisopropylpyrophosphoramide (iso-OMPA; Sigma, St.
biopsies of mice. We determined TrkA and TrkB genotypes by PCR Louis,MO).Sectionsfromknock-outandwild-typeanimalswerealways
amplification using, respectively, a common 5(cid:57) primer (5(cid:57)-GACCCTG- stainedinparallel.
CACTGTCGAGTTTGC-3(cid:57)) and either a 3(cid:57) primer for wild-type allele Immunocytochemistry.Perfusion,fixation,andpreparationofsections
(5(cid:57)-CGGACCTCAGTGTTGGAGAGCTGG-3(cid:57)) or a primer from the wereperformedasdescribedabove.Cryostatsections(14,20(cid:109)m)were
pgk-1 promoter of the neo cassette (5(cid:57)-GCTCCCGATTCGCA- mountedongelatin-coatedslides,driedatRT(for30min),andplacedin
GCGCATCG-3(cid:57)) and a common 5(cid:57) primer (5(cid:57)-TCGCGTAAAGAC- 0.1Mphosphatebuffer(PB),pH7.4.Nonspecificbindingswereblocked
GGAACATGATCC-3(cid:57)) and either a 3(cid:57) primer for the wild-type allele bypreincubationwith5%normalgoatserumand0.1%TritonX-100in
(5(cid:57)-AGACCTGATGAGTGGGTCGCC-3(cid:57)) or a 3(cid:57) primer from the PBfor1hratRT.Sectionswereimmunostainedasfollows:(1)incuba-
pgk-1 promoter of the neo cassette (5(cid:57)-GATGTGGAATGTGT- tionwithprimaryantiserum(fordetails,seeTable1)anddilutedinPB
GCGAGGCC-3(cid:57)).ThePCRproductwasanalyzedona1.5%agarosegel. (containing2%normalgoatserum,1%bovineserumalbumin,and0.1%
InsituhybridizationofTrkAmRNA.Wild-typemice(P6,n(cid:53)2;P12, TritonX-100)for12hratRT;(2)incubationwithCy3-conjugatedgoat
n (cid:53) 2; adult, n (cid:53) 2) were anesthetized and perfused with 4% parafor- anti-rabbitIgG(Dianova,Hamburg,Germany)anddiluted1:2000inPB
maldehyde(PFA).Adrenalglandsweredissected,post-fixed(12hr),and for1hratRT.Controlswereperformedbyusingrabbitnormalserumor
processed for paraffin embedding. Deparaffinized sections (7 (cid:109)m) were by omitting the respective antiserum. Finally, all sections were rinsed
rehydratedandwashedin0.83%NaClandinPBS.Thensectionswere threetimesinPB,dried,andembeddedinKaiser’sglycerolgelatin.
post-fixedfor10minin4%PFA,washedtwotimesinPBS,andincubated Westernblotanalysis.Freshadrenalglandsfromwild-type((cid:49)/(cid:49)),TrkA
for 30 min with proteinase K (20 (cid:109)g/ml in 50 mM Tris/0.5 M EDTA), ((cid:49)/(cid:50);(cid:50)/(cid:50)),andTrkB((cid:50)/(cid:50))animals(P6;pooledfromtwoanimalsper
followed by washes in PBS/0.2% glycine and PBS. Sections again were genotype)werehomogenizedinthepresenceofdetergent,andproteins
post-fixedfor10minin4%PFA,rinsedinPBSanddistilledwater,and wereseparatedona7.5%acrylamide-containingSDS-gel(2(cid:109)g/lane)in
thereafter incubated for 10 min in 1.3% triethanolamine/0.31% acetic aminichamber(Bio-Rad,Richmond,CA).Aftertransfertonitrocellu-
anhydratein0.05NHCl.Finally,slideswerewashedinPBSand0.83% lose, the proteins were incubated with a monoclonal AChE antibody
NaCl,dehydrated,andair-dried. (diluted 1:2500; Transduction Laboratories, Lexington, KY) overnight.
Hybridization was performed in 50% formamid, 0.3 M NaCl, 20 mM TheprimaryantibodywasvisualizedviaamouseVectastainkit(Vector
Tris,pH7.5,5mMEDTA,10%dextransulfate,1(cid:51)Denhardt’ssolution, Laboratories, Burlingame, CA). Finally, the bands were analyzed by
0.5mg/mltotalyeasttRNA,and10mMdithiothreitol(DTT)with1(cid:51)107 computerdensitometry(ImageQuant).
cpm of 35S-UTP-labeled cRNA probe, which was transcribed with T7 Determination of AChE activity. AChE activity was determined in
polymerase from pRB6, a pBS KS(cid:50) carrying a 398 bp insert from the homogenatesoffreshadrenalglands(pooledfromthreeanimals)andthe
TrkAreceptor.Sectionswerehybridizedovernightat60(cid:56)Cinahumidi- thoracic spinal cord by a microtiter plate-adapted modification of the
fiedchamber.Onthenextdayslideswerewashedfor1hrat55(cid:56)Cin2(cid:51) Ellmanmethod(Ellmanetal.,1961)at405nm(20min).Thesubstrate
SSC,50%formamid,and10mMDTTandthenfor1hrat55(cid:56)Cin2(cid:51) concentration for acetylthiocholine iodide was 1.5 mM. To inhibit any
SSC,50%formamid,and10mMDTT.Subsequently,slideswererinsed butyrylcholineesteraseactivity,wedeterminedtheAChEactivitiesinthe
threetimesfor10minat37(cid:56)CinNTEbuffer(0.5MNaCl,10mMTris, presence of 0.1 mM iso-OMPA. The protein content in each of the
and5mMEDTA)andthenincubatedfor30minat37(cid:56)Cwith20(cid:109)g/ml homogenates was quantified according to the Bradford method (1976),
RNaseAinNTEbuffer.Aftera15minprocessinginNTEbuffer,slides usingbovineserumalbuminasastandard.Resultsweregivenasmean
werewashedfor1hrat55(cid:56)Cin2(cid:51)SSC,50%formamid,and10mMDTT values(cid:54)SEMandtestedforsignificancebyStudent’sttest.
and then for 15 min at room temperature (RT) in 0.2(cid:51) SSC. After Determinationsofcatecholamines.Catecholaminesofsingleadrenalglands
dehydration,sectionswereair-dried,dippedinKodakNTB-2emulsion (P6)werequantifiedbyhighperformanceliquidchromatography(HPLC)
(diluted1:1inwater),exposedfor4weeksat4(cid:56)C,developed,fixed,and and electrochemical detection essentially as described by Mu¨ller and Un-
counterstainedwithhematoxylin. sicker(1981).TheamountsofcatecholamineswereaveragedandtheSEM
AChEhistochemistry.Stainingwasperformedaccordingtoamodifica- wascalculated.StatisticalsignificancewasdeterminedbyStudent’sttest.
tionof thedirectcoloringthiocholinemethodofKarnovskyand Roots Cellcounts.Nissl-stainedthoracicspinalcordsections(TrkA(cid:50)/(cid:50),n(cid:53)
(1964)forhistochemicaldetectionofAChEactivity(Andr¨aandLojda, 4;TrkB(cid:50)/(cid:50),n(cid:53)3;wild-type(cid:49)/(cid:49),n(cid:53)3)wereusedfordetermining
1986).Adrenalglandsandthefulllengthofthethoracicspinalcordwere numbers of sympathetic preganglionic neurons (100 adjacent cross sec-
removedfromperfusedTrkA((cid:50)/(cid:50)),TrkB((cid:50)/(cid:50)),andwild-type((cid:49)/(cid:49)) tions per animal, 20 (cid:109)m, segments T8–T10). To identify sympathetic
animals (4% PFA in phosphate buffer, pH 7.4), cryoprotected (30% preganglionicneuronsreliably,wecounterstainedNissl-stainedsections
sucrose),frozenondryice,andcutinto20-(cid:109)m-thicksections.Sections weaklybyAChEhistochemistryasdescribedabove.Onlyneuronswitha
were mounted on slides and stained for 1 hr at 37(cid:56)C in the following clearly visible nucleus were counted, and the total number of labeled
solution (60 ml): 30.0 mg of acetylthiocholine iodide (Serva Feinbio- neurons was estimated according to Abercrombie’s formula (Konigs-
chemica,Heidelberg,Germany),44.4mlof0.1MTris-maleatebuffer,pH mark, 1970). Results were given as mean values SD and tested for
5.0(containing0.1%TritonX-100),6.0mlof0.4Msodiumcitrate,6.0ml statisticalsignificancebyStudent’sttest.
Schoberetal.•TrkA-MediatedRegulationofAChEActivity J.Neurosci.,February1,1997,17(3):891–903 893
Figure1. InsituhybridizationofTrkAmRNAusinganantisenseprobe(a,c,e)andasensecontrol(b,d,f)intheP6,P12,andadultmouseadrenal
glandsshowsspecificlabelingoftheadrenalmedulla(am),whichisparticularlyprominentoverclustersofchromaffincells.Thesurroundingcortex(ac)
showsbackgroundlabeling.Scalebars,200(cid:109)m.
RESULTS ing sense controls in mouse adrenal glands at postnatal ages P6
TrkA is expressed in the developing and adult mouse (Fig.1a,b),P12(Fig.1c,d),andintheadult(Fig.1e,f).Duringall
adrenalmedulla postnatalages,labelingwasconfinedtothecenterportionofthe
ToestablishthatNGFresponsivenessandhigh-affinitybindingof gland, consistent with the expression of TrkA within the adrenal
NGFtochromaffincells(forreferences,seeDiscussion)reflectan medulla and its chromaffin cells. No signal was detectable at P0
expression of TrkA, we performed in situ hybridization studies. (datanotshown).AdrenalchromaffincellsdidnotexpressTrkB
Figure1showsthelocalizationofTrkAmRNAandcorrespond- attheseagesinmice(datanotshown).
894 J.Neurosci.,February1,1997,17(3):891–903 Schoberetal.•TrkA-MediatedRegulationofAChEActivity
Figure2. LocalizationofAChEactivitybyhistochemistryintheadrenalgland.AtP3(a,b),therearenodifferencesinAChEactivityandlocalization
betweenwild-type((cid:49)/(cid:49))andTrkAknock-out((cid:50)/(cid:50))animals.Thegrossmorphologyoftheadrenalglandapparentlyisunchangedintheknock-outs.At
P7(c,d)andP12(e,f),AChEactivityisreducedinTrkA-deficientmice(d,f),ascomparedwithwild-type(c,e)animals.Scalebars,200(cid:109)m.
AChE histochemical staining of the adrenal medulla is of wild-type mice ((cid:49)/(cid:49); Fig. 2a,c,e) and animals deficient for
reduced in TrkA ((cid:50)/(cid:50))mice TrkA ((cid:50)/(cid:50); Fig. 2b,d,f). At postnatal days P0 (data not shown)
Figure2illustratesthetemporaldevelopmentofhistochemically andP3(Fig.2a,b),therewerenodetectabledifferencesinAChE
demonstrableAChEactivityintheearlypostnataladrenalgland staining between wild-type and TrkA knock-out mice. At these
Schoberetal.•TrkA-MediatedRegulationofAChEActivity J.Neurosci.,February1,1997,17(3):891–903 895
Figure 3. Localization of AChE activ-
ity by histochemistry in thoracic spinal
cord. In TrkA-deficient mice (b), there
isastrongreductionofAChEactivityin
autonomicnucleiaswellasinthesuper-
ficiallayersofthedorsalhorn(DH),as
compared with the wild-type (a). IML,
Intermediolateral column; NC, nucleus
centralis; VH, ventral horn. c, Longitu-
dinal section of thoracic spinal cord
(T7–T10) showing AChE-positive
preganglionic sympathetic neurons lo-
cated in IML and NC of a wild-type
animal.d,Boththenumberofreactive
neuronsandtheirAChEactivityarede-
creaseddramaticallyinTrkAknock-out
mice.Scalebars,200(cid:109)m.
earlyages,AChEactivitywasveryweakbutclearlyconfinedtothe and specific for this neuron population, we studied the thoracic
adrenalmedullaandnervefiberstraversingthecortex.(Fig.2a,b). spinalcord,wheretheseneuronsarelocatedintheIMLcolumn
In wild-type animals (Fig. 2c,e) AChE activity clearly was in- (Strack et al., 1988). Figure 3a–d shows that within the thoracic
creasedatP7andP12,ascomparedwithP3,andassociatedwith spinal cord of TrkA ((cid:50)/(cid:50)) mice (P12) AChE staining intensity
cells,fiberbundles,anddelicatestrandsofaxonsinnervatingthe clearlywasreduced,ascomparedwithwild-typespecimens.This
adrenal medulla. In TrkA ((cid:50)/(cid:50)) mice a dramatic decrease in reduction was very pronounced in cell bodies of preganglionic
AChE histochemical staining became apparent at P7 (Fig. 2d), sympathetic neurons located in the IML column and in nerve
resultinginanalmostcompletelossofadrenalAChEstainingin fibers within the dorsal horn. AChE staining in motoneurons
P12animals(Fig.2f). appearedtobeunaffected.Asintheadrenalmedulla,alterations
in spinal cord AChE staining became apparent at P7 and pro-
AChE histochemical staining of preganglionic
gressedtowardP12(datanotshown).
sympathetic neurons, but not motoneurons, in the
spinal cord is reduced in TrkA ((cid:50)/(cid:50)) mice AChE histochemical staining is unaltered in adrenal
ToinvestigatewhetherthereductioninAChEactivityofadrenal gland and spinal cord IML neurons of TrkB ((cid:50)/(cid:50)) mice
medullarynervefibersinTrkA((cid:50)/(cid:50))micewasaccompaniedby To exclude that alterations in AChE activity seen in adrenal
adecreaseofAChEactivitywithintheperikaryaoftheseneurons gland and spinal cord of TrkA-deficient mice were unspecific,
896 J.Neurosci.,February1,1997,17(3):891–903 Schoberetal.•TrkA-MediatedRegulationofAChEActivity
Figure 4. AChE histochemical staining in adrenal medulla (a, b) and thoracic spinal cord (T7–T10; c, d) of TrkB-deficient mice, as compared with
wild-typelittermates.AtP12thereisnodifferenceinAChEactivityandlocalizationdetectablebetweenTrkBknock-outs(b,d)andwild-typecontrols
(a,c).IML,Intermediolateralcolumn;NC,nucleuscentralis.Scalebars,200(cid:109)m.
resulting from severe illness of these animals, we investigated tide Y (NPY) immunoreactivity was present in most, if not all,
TrkB ((cid:50)/(cid:50)) mice that also died during the early postnatal adrenal chromaffin cells of wild-type mice as well TrkA- and
period. Patterns and intensities of AChE staining in adrenal TrkB-deficientanimalsatP0,P7,andP12.Therewasaminor,yet
medullary nerve fibers and thoracic spinal cord IML neurons inconsistent, increase in the intensity of immunofluorescence in
were undistinguishable in TrkB ((cid:50)/(cid:50)) and wild-type ((cid:49)/(cid:49)) the knock-out animals (Fig. 6c,d). Subpopulations of chromaffin
mice(Fig.4a–d).ThisresultsuggeststhatTrkA,butnotTrkB, cells displayed immunoreactivities for somatostatin, met-
is involved in the postnatal regulation of AChE activity in enkephalin, and galanin, which were weak or nonexistent at P0
preganglionic sympathetic neurons of the spinal cord. and P6 but clearly apparent at P12, with no notable differences
betweenwild-typeandknock-outanimals.Immunoreactivitiesfor
Quantitative determination of specific AChE activity
synaptophysinandtheadhesionmoleculeL1wereassociatedwith
reveals a pronounced reduction in adrenal gland and
thoracic spinal cord homogenates of TrkA ((cid:50)/(cid:50)) mice, fibers and varicosities surrounding chromaffin cells and cell clus-
but not in TrkB-deficient mice ters.Boththedistributionalpatternsandfluorescenceintensities
were unaltered in the knock-out animals. Likewise, patterns and
Quantitative analysis of AChE activity in homogenates from
intensities of the immunoreactivities for tyrosine hydroxylase
whole adrenal glands provided evidence that the decrease in
AChE staining intensity seen in TrkA ((cid:50)/(cid:50)) mice was, in fact, (TH) and phenylethanolamine N-methyltransferase (PNMT),
chromogranin B, secretogranin II, and the vesicular monoamine
attributable to a substantial reduction in AChE activity (P7;
(cid:50)62% relative to wild-type littermates, Fig. 5a). In the thoracic transporters I and II (VMAT-1, VMAT-2) were not affected by
theTrkAandTrkBdeficits.
spinalcordofthesameanimals,a40%decreaseinspecificAChE
activity could be demonstrated (Fig. 5b). In adrenal glands and
Immunocytochemical staining of AChE is unaltered in
thoracic spinal cords of TrkB knock-outs, the specific AChE adrenal medullae of TrkA ((cid:50)/(cid:50))mice
activitywasnotalteredsignificantly(Fig.5a,b).
Given the lack of alterations in any of the above axonal and neu-
Chromaffin cell-associated markers detected by roendocrine markers, we investigated whether changes in AChE
immunocytochemistry are not changed overtly in TrkA staining using enzyme activity as an indicator were reflected in
((cid:50)/(cid:50)) and TrkB ((cid:50)/(cid:50)) mice alterations of immunocytochemically demonstrable AChE protein.
Tomonitorotherputativedeficitsofadrenalmedullarydevelop- As shown in Figure 6a,b, the polyclonal antibody to AChE clearly
ment in TrkA knock-outs, we used immunocytochemistry with revealedthelocalizationofAChEinnervefiberssupplyingadrenal
antibodies to a number of markers associated with chromaffin medullarycellswithoutshowinganydifferencesintheintensitiesof
cellsandtheirpreganglionicnervefibers(seeTable2).Neuropep- the immunocytochemical staining. These results suggest that the
Schoberetal.•TrkA-MediatedRegulationofAChEActivity J.Neurosci.,February1,1997,17(3):891–903 897
Table2.Summaryofsemiquantitativedatarevealedby
immunocytochemistry
Wild- TrkA TrkB
Structurelabeled type ((cid:50)/(cid:50)) ((cid:50)/(cid:50))
Neuropeptides
NeuropeptideY Chromaffincellsub- (cid:49)(cid:49) (cid:49)(cid:49)(cid:49) (cid:49)(cid:49)(cid:49)
populations(2)
Somatostatin Chromaffincellsub- (cid:49) (cid:49) (cid:49)
population
Met-enkephalin Chromaffincellsub- (cid:49) (cid:49) (cid:49)
population
Galanin Chromaffincellsub- (cid:49)(cid:49) (cid:49)(cid:49) (cid:49)(cid:49)
population
Innervation
Synaptophysin Fiberinnervation (cid:49)(cid:49) (cid:49) (cid:49)
L1 Fiberinnervationand (cid:49)(cid:49) (cid:49)(cid:49) (cid:49)(cid:49)
extracellularlabeling
Catecholaminesyn-
thesizingenzymes
TH Chromaffincellsub- (cid:49)(cid:49)(cid:49) (cid:49)(cid:49)(cid:49) (cid:49)(cid:49)(cid:49)
populations(2)
PNMT Chromaffincellsub- (cid:49)(cid:49) (cid:49)(cid:49) (cid:49)(cid:49)
population
Vesicular
ChromograninB Chromaffincellsub- (cid:49)(cid:49) (cid:49) (cid:49)
population
SecretograninII Chromaffincellsub- (cid:49)(cid:49) (cid:49)(cid:49) (cid:49)(cid:49)
population
VMAT-1 Allchromaffincells (cid:49) (cid:49) (cid:49)
VMAT-2 Chromaffincellsub- (cid:49) (cid:49) (cid:49)
population
*Denotestheapparentdegreeoflabeling:(cid:49)(cid:49)(cid:49)strong,(cid:49)(cid:49)moderate,(cid:49)weak.
we performed Western blot analysis. As shown in Figure 7, the
authentic68kDabandofAChEisequallyprominentinhomog-
enatesfromwild-type,TrkAhetero-andhomozygotes,andTrkB
homozygote mice. Densitometric analysis revealed a 3.2% de-
crease of the band from TrkA ((cid:50)/(cid:50)) mice, as compared with
wild-typelittermates.
Cell counts reveal a reduction in IML neuron numbers
in TrkA-deficient, but not in TrkB-deficient,mice
Cell counts performed on double-stained (Nissl/AChE) serial
cryosections (100 adjacent cross sections/animal) through tho-
racicspinalcordlevelsT8–T10ofTrkA-deficientmicerevealeda
Figure5. BiochemicaldeterminationofAChEactivityinhomogenates. reduction in IML neuron numbers by 41.5%, as compared with
At P7, specific AChE activity is decreased significantly ((cid:50)62%) in the
wild-type controls (Figs. 8, 9). In contrast, the number of IML
adrenalgland(a)ofTrkAknock-outanimals((cid:49)/(cid:49),n(cid:53)3;(cid:50)/(cid:50),n(cid:53)3;
*p(cid:46)0.05).Inthoracicspinalcord(b),thereisa40%reductionofspecific neuronsinTrkB-deficientmicewasnotaffected(Figs.8,9).
AChE activity ((cid:49)/(cid:49), n (cid:53) 12; (cid:50)/(cid:50), n (cid:53) 9) relative to the wild-type. In
Catecholamines are reduced in adrenal glands of TrkA
contrast, levels of AChE activity of adrenal glands and spinal cords in
TrkBknock-outanimalsarenotalteredsignificantly.Errorbars,(cid:54)SEM. ((cid:50)/(cid:50))mice
To investigate whether the chronic loss of AChE activity in
TrkA-deficientanimalshadanimpactonthecatecholaminestor-
TrkA receptor knock-out clearly affects the activity of the enzyme,
age and secretion of chromaffin cells, we determined the cat-
without affecting the presence of preganglionic nerve fibers within
echolamines adrenaline and noradrenaline by HPLC–ampero-
theadrenalgland.Moreover,expressionoftheproteinandenzyme
metric detection. As shown in Figure 10, there was a significant
activityseemstobedifferentiallyregulated.
reductioninthemedullarylevelsofbothamines(noradrenaline,
Western blot analysis of AChEprotein (cid:50)63.5%; adrenaline, (cid:50)70.7%), probably because of the pro-
To support further the notion that AChE immunoreactivity in longed activation of catecholamine secretion by acetylcholine at
adrenalglandsofTrkA((cid:50)/(cid:50))animalswasprimarilyunchanged, reducedactivityofthehydrolyzingenzyme.
898 J.Neurosci.,February1,1997,17(3):891–903 Schoberetal.•TrkA-MediatedRegulationofAChEActivity
Figure6. AChE-immunoreactivenervefibers(a,b)andNPYimmunoreactivity(c,d)intheadrenalmedulla.AtP7(a,b),densityanddistribution
patternofAChE-immunostainedfibersarenotdistinguishableinwild-type(a)andTrkAknock-outmice(b).NPYimmunoreactivityinchromaffincells
isincreasedslightlyinknock-out,ascomparedwithwild-type,miceatP12.Scalebars,100(cid:109)m.
DISCUSSION glandandspinalcordofTrkA((cid:50)/(cid:50))micefailstocorrelatewith
The present results add a novel feature to the previously estab- an overt change in the immunocytochemically demonstrable
lished TrkA ((cid:50)/(cid:50)) phenotype, a severe deficit within adrenal AChEandAChEproteindetectableinWesternblots,suggesting
glandandpreganglionicsympatheticspinalcordneuronsregard- that AChE activity is compromised more severely than AChE
ing the activity of AChE, an enzyme with a well documented proteinexpression.Thisaddstothegrowingevidencethatapool
crucial role in terminating transmitter actions at cholinergic syn- ofinactiveAChEproteincanbeactivatedpost-translationallyin
apses (for review, see Massouli´e et al., 1993). The evidence that anenvironment-dependentmanner(forreview,seeMassouli´eet
the TrkA knock-out affects AChE activity is based on a specific al., 1993; Layer and Willbold, 1995). Uncompromised AChE
histochemical staining technique (Andr¨a and Lojda, 1986) and a protein expression in the adrenal gland of TrkA ((cid:50)/(cid:50)) mice is
quantitative photometric method for determining specific AChE also in accord with an unimpaired localization of synaptophysin
activity (Ellman et al., 1961) in homogenates. AChE staining withintheadrenalgland.
patternandintensityreflectthequantitativedistributionofAChE The neuroendocrine chromaffin cells of the adrenal medulla
activity, because there is a linear correlation between enzyme and sympathetic neurons share an ontogenetic origin from the
activity quantified photometrically and staining intensity (Andr¨a neural crest (cf. Unsicker, 1993) and many structural and func-
andvanDuijn,1985).ThedeficitinAChEactivityseeninadrenal tional features, including a prominent cholinergic innervation
Schoberetal.•TrkA-MediatedRegulationofAChEActivity J.Neurosci.,February1,1997,17(3):891–903 899
medulla do not die on NGF withdrawal by treatment with NGF
antibodies(Bodeetal.,1986).Theydorespond,however,toNGF
invitrowithneuriteoutgrowth(Unsickeretal.,1978;Doupeetal.,
1985),amoderateincreaseinsurvival(Unsickeretal.,1985a,b),
inductionofTH,andAChEactivity(Achesonetal.,1984;Mu¨ller
andUnsicker,1986).High-affinitybindingsitesforNGFonchro-
maffincells(Hofmannetal.,1987),TrkAmRNAinnewbornrat
adrenal medulla (Suter-Crazzolara et al., 1997), and localization
ofTrkAmRNAintheadultrat(Michaeletal.,1995)andmouse
adrenalmedulla(thisstudy)stronglyargueinfavorofTrkAbeing
expressed by chromaffin cells. The 62% reduction in adrenal
AChEofTrkA(cid:50)/(cid:50)mutantsshowninthepresentstudyprobably
can be attributed to reduced AChE activity in both the pregan-
glionicnervecellsandfibersaswellaschromaffincells(compare
Figs. 2, 3). AChE has been reported to appear in adrenal nerve
terminals and very few chromaffin cells of the rat around birth,
gradually increasing toward adulthood (Millar and Unsicker,
Figure7. Westernblotanalysisshowingimmunoreactive68kDaAChE 1981).ThissuggeststhatAChEactivityinnervefibersandchro-
proteininadrenalglandsofP6wild-type,TrkAhetero-andhomozygote,
maffincellsmaynothavereachedadultlevelsatP7andP12.
andTrkB-deficientanimals.
AChE-positive neurons within the IML column sending axons
to sympathetic ganglia and adrenal medulla have not been re-
ported to express TrkA. In fact, small interneurons, but not
autonomic preganglionic neurons within the rat spinal cord, are
immunoreactiveforTrkAandexpressTrkAmRNA(Michaelet
al., 1995; K. Huber, unpublished observation). Consistent with
this observation, NGF, in contrast to fibroblast growth factor-2,
ciliary neurotrophic factor, and transforming growth factor-(cid:98),
does not rescue IML neurons after ablation of one of their
prominent targets, the adrenal medulla (Blottner et al., 1989a,b,
1996). Nonetheless, cell counts of Nissl-stained neurons in the
IMLofthethoracicspinalcordbetweensegmentsT8andT10in
TrkA-deficient and wild-type mice show a (cid:46)40% reduction, re-
flectingneurondeathorshrinkage.Fromthesesegments25%of
the IML neurons are known to project to the adrenal medulla,
while the remaining 75% of the neurons project to prevertebral
(coeliac,aorticorenal,superiormesenteric,other)andparaverte-
bralsympatheticganglia(Stracketal.,1988;Blottneretal.,1996).
Our calculations, based on published cell counts and retrograde
tracings(Stracketal.,1988),indicatethatatleast65%oftheIML
neurons projecting to paravertebral ganglia could have disap-
pearedintheTrkAknock-outs(compareFig.11).Consistentwith
thisnotion,ithasbeenshowninchickembryosthatNGFappar-
Figure 8. Cell counts of serial transverse sections through spinal cord
entlyindirectlyregulatesthesurvivalofthesepreganglionicsym-
segments T8–T10 reveal a 41.5% reduction in IML neuron numbers in
TrkA ((cid:50)/(cid:50)), as compared with wild-type mice. In TrkB-deficient mice pathetic (the so-called Terni column) neurons within the spinal
numbersofIMLneuronsarenotaffected,p(cid:46)0.01. cordbyaffectingthesurvivaloftheirtargetcells,thepostgangli-
onic sympathetic neurons (Oppenheim et al., 1982). Thus, the
reduction of AChE activity in autonomic neurons within the
(CouplandandHolmes,1958;LewisandShute,1969;Millarand spinal cord of TrkA mutants is most likely attributable to a
Unsicker, 1981; Ahonen, 1991). Both pre- and postganglionic reductionofenzymeactivityinsparedIMLneuronsthatproject
noradrenergic sympathetic neurons, as well as chromaffin cells, totheadrenalmedullaandprevertebralganglia.
synthesizeandsecreteAChE(LewisandShute,1969;Mizobeand In contrast to postganglionic paravertebral sympathetic neu-
Livett, 1980, 1984; Millar and Unsicker, 1981; Hefti et al., 1982; rons,whichvirtuallyalldisappearinTrkAknock-outs(Smeyneet
Ahonen, 1991; Parker et al., 1993; Small et al., 1993). The func- al., 1994), many aspects of the structure and chemistry of the
tional implications of TrkA and NGF for the development and adrenalmedullaappearunchanged.Establishedmarkersforchro-
maintenance for each of these neural crest derivatives seem to maffin cells, including various neuropeptides (NPY, met-ENK,
diverge considerably. Both the recent targeted mutations of the somatostatin, galanin), markers of chromaffin granules (chromo-
TrkAandNGFgenes(Crowleyetal.,1994;Smeyneetal.,1994) granin B, secretogranin II), and vesicular amine transporters
and the early immunosympathectomy experiments (Angeletti et (VMAT-1/VMAT-2), are normally expressed and appear not to
al., 1972) support the essential physiological role of TrkA and bechangedovertly.Asinthewild-typemouse,allchromaffincells
retrogradely acting NGF to prevent ontogenetic death of the haveTHimmunoreactivity,and(cid:59)75%ofthemexpressPNMT.In
paravertebral sympathetic neurons [for review, cf. Snider (1994) additiontothereductionofAChEactivity,asignificantdecrease
andRushetal.(1995)].Incontrast,chromaffincellsoftheadrenal in the catecholamine content is the only other hallmark of the
900 J.Neurosci.,February1,1997,17(3):891–903 Schoberetal.•TrkA-MediatedRegulationofAChEActivity
Figure9. IllustrationofthequantitativedatapresentedinFigure7.Nissl-stainedsectionsfromspinalcordsegmentsT8–T10(a,c,e)andIML(b,d,
f).DH,Dorsalhorn;IML,intermediolateralcolumn;NC,nucleuscentralis;VH,ventralhorn.Scalebars:ac,e,200(cid:109)m;b,d,f,30(cid:109)m.
TrkA(cid:50)/(cid:50)adrenalmedullaryphenotype.Bothphenomenaprob- are densely innervated by sympathetic nerves (Shelton and
ably are causally linked in that loss of the acetylcholine- Reichardt,1984).Furthermore,mouseadrenalexplantssecretea
hydrolyzing enzyme probably accounts for a prolonged and neurotrophic activity into their culture medium, which can be
chronic activation of secretion of catecholamines, leading to a blockedbyNGFantibodies(Harper,1976).Takentogether,these
partialdepletionofcatecholaminesfromtheadrenalmedulla. datasuggestthattheadrenalglandinvivoisasourceofNGFand
Although a reduction of AChE activity in TrkA-expressing thatchromaffincellsmaybethetargetfortheadrenalNGF.
chromaffincellsmaybeacceptedreadilyasafeatureoftheTrkA InthespinalcordofTrkAmutants,AChEactivitywasnotonly
knock-out, the reduction in IML neurons lacking TrkA is more affected in the IML neurons but also with regard to AChE-
difficulttoexplain.WeassumethatthereductioninAChEactivity positive nerve fibers in the superficial and deeper layers of the
ofIMLneuronsislikelytobeasecond-ordereffect.Onepossible dorsalhorn.Thesefibersareintheirmajorityaxonsofdorsalroot
explanation might be that chromaffin cells provide IML neurons ganglionic(DRG)neurons,whicharevirtuallyallAChE-positive
with a signal, the expression of which in chromaffin cells can be (Gruberetal.,1971).ThedecreaseinAChEstaininginthedorsal
affectedbyaTrkA-mediatedmechanism.Themolecularnatureof horn of TrkA mutants is, therefore, likely to reflect the 70–90%
such a retrograde AChE-regulating messenger remains to be loss of DRG neurons and loss of small sensory afferents in the
elucidated. TrkAknock-out(Smeyneetal.,1994).
Several lines of evidence indicate that NGF is synthesized AChE activity in spinal cord somatic motoneurons apparently
withintheadrenalgland.NGFmRNAlevelsshownbyNorthern wasunalteredinTrkA((cid:50)/(cid:50)),aswellasinTrkB((cid:50)/(cid:50))mice,as
blottinginadultrabbitadrenalsamountto(cid:59)25%ofmRNAlevels compared with wild-type littermates. Somatic motoneurons ex-
inthespleenand(cid:59)20%ofheartatriumandventricle,allofwhich press TrkB, TrkC, and p75 neurotrophin receptors (Koliatsos et