Brauer, K

Grosche, J

Harkany, T

Hartig, W

Härtig, Wolfgang

Kacza, J

Luiten, PGM

Seeger, J

Varga, C

English

Cholinergic basal forebrain neurons (CBFN) expressing the low-affinity neurotrophin receptor p75 (p75(NTR)) were previously selectively labeled in vivo with carbocyanine 3 (Cy3)-tagged anti-p75(NTR), but the applied 192IgG-conjugates recognized p75(NTR) only in rat The antibody ME 20.4 raised against human p75(NTR) had been shown to cross-react with the receptor in monkey, raccoon, sheep, cat, dog, pig and rabbit. Hence, for in vivo labeling of rabbit CBFN in the present study, ME 20.4 was fluorochromated with Cy3-N-hydroxysuccinimide ester and purified Cy3-ME 20.4 was injected intracerebroventricularly. Two days post-injection, clusters of Cy3-ME 20.4 were found in CBFN displaying choline acetyltransferase-immunoreactivity. Following photoconversion, electron microscopy revealed fluorochromated antibodies in secondary lysosomes. In conclusion, Cy3-ME 20.4 might become an appropriate marker for CBFN in live and fixed tissues of various mammalian species.</p

Proceedings - University of Groningen

   University of GroningenIn vivo labeling of rabbit cholinergic basal forebrain neurons with fluorochromated antibodiesHartig, W; Varga, C; Kacza, J; Grosche, J; Seeger, J; Luiten, PGM; Brauer, K; Harkany, T;Härtig, WolfgangPublished in:NeuroreportIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2002Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Hartig, W., Varga, C., Kacza, J., Grosche, J., Seeger, J., Luiten, PGM., Brauer, K., Harkany, T., & Härtig,W. (2002). In vivo labeling of rabbit cholinergic basal forebrain neurons with fluorochromated antibodies.Neuroreport, 13(11), 1395-1398.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 11-10-2022In vivo labeling of rabbit cholinergic basal forebrainneurons with £uorochromated antibodiesWolfgang H	rtig,CA Csaba Varga, Johannes Kacza,1 Jens Grosche, Johannes Seeger,1 Paul G. M. Luiten,2Kurt Brauer and Tibor Harkany2,3Paul Flechsig Institute for Brain Research,University of Leipzig, Jahnallee 59,D-04109 Leipzig; 1Department of Anatomy,Histology and Embryology,University of Leipzig, An denTierkliniken 43,D-04103 Leipzig,Germany; 2Department of Molecular Neurobiology,University of Groningen,Kerklaan 30,NL-9750 AAHaren,The Netherlands; 3Department of Medical Chemistry, Faculty of Medicine,University of Szeged, Szeged,HungaryCACorresponding AuthorReceived 2 May 2002; accepted 9 May 2002Cholinergic basal forebrain neurons (CBFN) expressing the low-af-¢nity neurotrophin receptor p75 (p75NTR) were previously selec-tively labeled in vivo with carbocyanine 3 (Cy3)-tagged anti-p75NTR, but the applied192IgG-conjugates recognizedp75NTR onlyin rat.The antibodyME 20.4 raised against humanp75NTR hadbeenshown to cross-reactwith thereceptor inmonkey, raccoon, sheep,cat, dog, pig andrabbit.Hence, for invivo labeling of rabbit CBFNinthe present study, ME 20.4 was £uorochromated with Cy3-N-hy-droxysuccinimide ester and puri¢ed Cy3-ME 20.4 was injectedintracerebroventricularly. Two days post-injection, clusters ofCy3-ME 20.4 were found in CBFN displaying choline acetyltrans-ferase-immunoreactivity. Following photoconversion, electronmicroscopy revealed £uorochromated antibodies in secondarylysosomes. In conclusion,Cy3-ME 20.4 might become an appropri-atemarker forCBFNin live and ¢xed tissues of variousmammalianspecies. NeuroReport 13:1395^1398 c 2002 Lippincott Williams &Wilkins.Key words: Choline acetyltransferase; Cholinergic system; Low-a⁄nity neurotrophin receptor; Oxygen-enrichedphotoconversionINTRODUCTIONSevere damage to cholinergic neurons of the basal forebrain(BFB) is a major neuropathological hallmark of Alzheimer’sdisease, and this has prompted investigations focused onthis brain region in various mammalian species [1].Neuroanatomical studies have achieved the identificationof cholinergic nerve cells by the detection of their markerenzyme choline acetyltransferase (ChAT, EC 3.2.1.6) as wellas of vesicular acetylcholine transporters. An additionalselective marker of cholinergic BFB neurons is the low-affinity neurotrophin receptor p75 (p75NTR), as was demon-strated in the rat [2], raccoon [3], monkey [4] and human [5].Targeting of p75NTR led to the development of cholinergicimmunolesion models. In particular, the in vivo applicationof antibodies directed against an extracellular epitope of ratp75NTR and conjugated to the ribosome-inactivating toxinsaporin (192IgG-saporin) caused selective lesions of choli-nergic projection neurons in the rat BFB [6].A further derivative of 192IgG, namely its conjugate withthe red fluorochrome carbocyanine 3 (Cy3-192IgG), wasintroduced as a tool for the selective labeling of ratcholinergic, p75NTR-bearing neurons both in vivo [7,8] andin vitro [9]. 192IgG-conjugates are commonly infusedintracerebroventricularly and bind to p75NTR on cholinergicterminals, become internalized as ligand/receptor com-plexes and are transported retrogradely to the perikarya ofcholinergic BFB neurons [6]. Internalized Cy3-192IgGmolecules form clusters in secondary lysosomes [8].Whereas ribosome inactivation by saporin causes neuronaldeath [6,10], Wu et al. [11] demonstrated that pre-labeling ofcholinergic neurons with Cy3-192IgG does not alter theirelectrophysiological and pharmacological properties in exvivo brain slice preparations containing the medial septum/diagonal band complex (MSDB). Concordantly, identifica-tion of cholinergic nerve cells with Cy3-192IgG in brainslices led to new insights in the synaptic organization ofneurons in the MSDB with implications for learning andmemory [11,12].However, 192IgG conjugates recognize p75NTR only in therat precluding their application in other mammalian species(e.g. monkeys) with a BFB chemoarchitecture resemblingthat in humans [13]. In contrast, the monoclonal antibodyME 20.4 raised against an extracellular epitope of the humanp75NTR [14] was demonstrated to cross-react with p75NTRalso in monkey, raccoon [3], rabbit, pig, dog and cat [15].Accordingly, the ME 20.4-saporin conjugate was shown tobe a selective cholinotoxin in the common marmoset [16],rhesus monkey [17], rabbit [18] and sheep [19].To overcome the restriction of in vivo labeling in the rat,the present study investigated whether Cy3-ME 20.4 can beemployed as a marker of living cholinergic neurons in awide range of mammalian species expressing analogues ofthe human p75NTR. Encouraged by recent data on ME 20.4-saporin-induced immunolesions of cholinergic cells and0959-4965c LippincottWilliams &Wilkins Vol 13 No 11 7 August 2002 1395NEUROCHEMISTRY, BASIC ANDCLINICAL NEUROREPORTsubsequent b-amyloid deposition in the rabbit brain [18], thepotential of Cy3-ME 20.4 as a label for cholinergic nervecells was exemplified in studies on BFB neurons of thisanimal species.MATERIALS AND METHODSPreparation of Cy3-ME 20.4: Cy3-ME 20.4 was preparedby coupling the antibody with a chemically activated esterof Cy3 under reaction conditions proved to maintain theantigen-binding properties of ME 20.4 [20]. Four hundredmicrograms of ME 20.4 (pure IgG; AB-N07, AdvancedTargeting Systems, San Diego, CA, USA) were reacted witha monofunctional Cy3-N-hydroxysuccinimide ester (Anti-body Cy3 Labeling Kit, Amersham Pharmacia Biotech,Freiburg, Germany) according to the instruction of themanufacturer at pH 9.3 for 1 h, but with doubled dye:pro-tein ratio. Thereafter, the reaction mixture was dialyzedagainst phosphate-buffered saline (3  4 h) and the con-centration of Cy3-ME 20.4 was adjusted to approximately0.5 mg/ml prior its use for intracerebroventricular (i.c.v.)injections.Surgical procedure: New Zealand white rabbits of eithersex (n¼ 5; 2.3–5.1 kg; 4–21 months of age) were used in thisstudy. The rabbits were anesthetized with a mixture ofketamine (50 mg/kg; Exalgon, Merck, Hallbergmoos, Ger-many) and xylazine (4 mg/kg; Rompun, Bayer AG, Lever-kusen, Germany), and their heads were mounted in astereotaxic frame. Two animals received unilateral i.c.v.injections, and three animals were injected bilaterally with16ml Cy3-ME 20.4 at an infusion speed of 0.5 ml/min with aHamilton microsyringe (Hamilton, Bonaduz, Switzerland).Injection co-ordinates were applied according to Beach et al.[18] at AP¼ 0.0 mm, L¼ 2.2 mm and DV¼ 9.0/7.5 mmrelative to bregma. All efforts were made to minimizeanimal suffering throughout the experiments includingadministration of the analgetic Metamizol (150 mg/kg;Berlosin, Berlin-Chemie, Germany) immediately after op-eration. The antibiotic Enrofloxacin (10 mg/kg daily; Baytril,Bayer AG) was given orally throughout the post-injectionperiod. The care and treatment of the animals were inaccordance with the European Communities Council Direc-tive (86/609/EEC) and approved by the Laboratory AnimalCare and Use Committee of the University of Leipzig (TVV-Nr.15/00).Immunocytochemistry and electron microscopy: Two dayspost-injection rabbits were transcardially perfused with 4%paraformaldehyde containing either 0.5% glutardialdehyde(three animals) or 0.1% glutardialdehyde (two animals) in0.1 M phosphate-buffer (pH 7.4). Whole brains wereremoved from the skulls, divided into fore- and hindbrainregions and post-fixed overnight in 4% paraformaldehyde.Two forebrains were coronally sectioned on a vibratome at50mm and the remaining tissue was cryoprotected and cutat 30 mm on a cryostat microtome. Sections spanning theMSDB and the anterior subdivision of the nucleus basalismagnocellularis were washed with Tris-buffered saline(TBS, 0.1 M, pH 7.4), rinsed in distilled water, mounted,air-dried and coverslipped with Entellan (in toluene; Merck,Darmstadt, Germany).To determine the specificity of in vivo labeling, Cy3-ME20.4 pre-labeled sections were applied to the concomitantimmunolabeling of ChAT and the calcium-binding proteincalretinin. Non-specific binding sites for the immunorea-gents were primarily blocked with 5% normal donkeyserum in 0.1 M Tris-buffered saline (TBS) containing 0.3%Triton X-100 for 1 h. Subsequently, the sections wereincubated with a mixture of affinity-purified goat anti-ChAT (1:25; AB144P, Chemicon International; Hofheim,Germany) and rabbit anti-calretinin (1:300; AB149, Chemi-con) diluted in the blocking solution for 16 h. Next,rinsed sections were reacted with a cocktail of Cy2-taggeddonkey anti-goat IgG and Cy5-conjugated donkey anti-rabbit IgG (20 mg/ml; Dianova, Hamburg, Germany) dilutedin TBS containing 2% bovine serum albumin for 1 h.Omission of the primary antibodies in control experimentsled to the absence of any cellular Cy2- and Cy5-labeling.After the cytochemical procedures the sections were rinsed,mounted, air-dried and coverslipped in Entellan. Fluores-cence microscopy was performed using an Axioplanmicroscope (Zeiss, Oberkochen, Germany) and selectedsections were inspected with a Zeiss 510 confocal laser-scanning microscope.To quench the autofluorescence caused by lipophiliclipofuscin-like compounds in sections from 21-month-oldrabbits, tissue was treated with Sudan Black B according toSchnell et al. [21] but prior to the immunofluorescencelabeling of cholinergic neurons. All Sudan Black B-pre-treated sections were embedded in glycerol/gelatin (Sigma,Deisenhofen, Germany).The subcellular distribution of Cy3-ME 20.4 was deter-mined following photoconversion of Cy3-ME 20.4 into anelectron-dense reaction product applying oxygen-enrichedphotoconversion in a closed conversion chamber as de-scribed previously [8]. Specimens were analyzed with aZeiss 900 electron microscope.RESULTSTwo days following the i.c.v. infusion of Cy3-ME 20.4 therewas strong punctate labeling in many cells of the MSDB(Fig. 1) that became less intense in the nucleus basalismagnocellularis. This staining pattern was observed irre-spective of the concentration of glutardialdehyde in thefixative. In comparison to rabbits unilaterally infused withCy2-ME 20.4, bilaterally injected animals appeared tocontain more in vivo-labeled nerve cells in both hemi-spheres.In vivo labeling of Cy3-ME 20.4 was exclusively found incholinergic neurons, as revealed by their immunoreactivityfor ChAT (Fig. 1a) and p75NTR (not shown). In contrast,in vivo labeling was never observed in non-cholinergic,calretinin-immunoreactive nerve cells (Fig. 1b) that areknown to be intermingled with cholinergic neurons in theborder zone of the medial and lateral septum [22] and inmagnocellular, parvalbumin-containing GABAergic BFBneurons (not shown).It is noteworthy that treatment of the sections from 21-month-old rabbits with Sudan Black B quenched theautofluorescence caused by lipophilic lipofuscin-like com-pounds. Importantly, Sudan Black B treatment only slightlydiminished the intensity of in vivo labeling with hydrophilic1396 Vol 13 No 11 7 August 2002NEUROREPORT W.HHRTIG ETAL.Fig. 1. The confocal laser-scanningmicrograph (a) shows red punctate Cy3-ME 20.4 in vivo labeling in cholinergic neurons of the rabbit medial septum.Selectivity of Cy3-ME 20.4 for cholinergic neurons was demonstrated by co-localizationwith choline acetyltransferase (ChAT) that appears green (Cy2).(b) Merged carbocyanine triple £uorescence staining of Cy3-ME 20.4 (red clusters) and immunoreactivities for ChAT (Cy2, green) and calretinin (Cy5,color-coded in blue) in the border zone between themedial and lateral septum con¢rmed the speci¢city of in vivo labeling for cholinergic neurons and itsabsence in non-cholinergic, calretinin-containing nerve cells. Bar¼ 25mm.Fig. 2. Electron micrographs of cholinergic neurons in the medial septum (a) and, at higher magni¢cation, in the diagonal band of Broca (b) followingphotoconversion of internalized Cy3-ME 20.4.The in vivo label was predominantly present in secondary lysosomes (arrows) after two days of survival. Incomparison to thephotoconvertedmaterial, non-labeledprimary lysosomes (arrowheads) are di¡erent in their size, shape and electron density. Prior toprint, the brightness and contrast of the electron micrographic images were digitally adjusted and shading correction was performed to reduce thee¡ects of uneven illumination. Bar¼ 500nm (a), 250 nm (b).Vol 13 No 11 7 August 2002 1397INVIVOLABELLINGWITHCY3-ME 20.4 NEUROREPORTCy3 and of the subsequently performed Cy2-immunostain-ing of cholinergic neurons.Electron microscopic analysis elucidated that Cy3-ME20.4 was predominantly located intracellularly in secondarylysosomes (Fig. 2a,b).DISCUSSIONThe granular cellular labeling of neurons in the rabbit BFBwith i.c.v. applied Cy3-ME 20.4 resembled that of Cy3-192IgGin the rat BFB [7]. A subpopulation of cholinergic neurons inthe MSDB was unlabeled by Cy3-ME 20.4 that also parallelsthe incomplete Cy3-192IgG labeling in the rat [7,11]. Recently,the counting of Cy3-192IgG-positive cells in the rat BFBrevealed a medial septumhorizontal limb of the diagonalband44nucleus basalis magnocellularis gradient of labelingefficacy [23]. An apparently similar gradient was observedafter the in vivo labeling with Cy3-ME 20.4; that, however,remains to be quantified. Future studies should also elucidatewhether the in vivo labeling with Cy3-ME 20.4 is long-termstable or undergoes gradual clearance from the neurons, aswas previously shown for Cy3-192IgG in the rat [8].The predominant location of Cy3-ME 20.4 in secondarylysosomes, as revealed by electron microscopy, is identicalto the ultrastructural localization of Cy3-192IgG in rat basalforebrain neurons [8]. This finding also corresponds withthe observation that lysosomes are preferred targets forinternalized nerve growth factor [24], the natural ligand ofp75NTR, which might be mimicked by fluorochromatedantibodies raised against p75NTR.Whereas the quenching of autofluorescence with SudanBlack B in the present work hardly affected the intensity ofin vivo labeling and subsequent immunofluorescence stain-ing, strong autofluorescence might interfere with theidentification of cholinergic neurons with Cy3-ME 20.4 inlive brain slices originating from aged animals.Our present findings suggest the potential use of Cy3-ME20.4 in future neuropharmacological studies targeting thecholinergic system of higher mammals. Such investigationsare also emphasized by the accelerated gathering ofpharmacological data based on the in vivo labeling of ratcholinergic basal forebrain neurons [11,12]. In particular,these studies showed that muscarinic receptor antagonistsinduce memory impairment due to a decrease of septo-hippocampal GABA release, instead of the previouslyassumed diminished hippocampal acetylcholine release,and confirmed neuroanatomical data on collaterals ofcholinergic neurons contacting parvalbumin-immunoposi-tive GABAergic neurons within the MSDB [25].CONCLUSIONThe high interspecies variability of the chemical nature,structure and topography of the cholinergic components inmagnocellular BFB nuclei hampers the development ofgeneral functional concepts based on investigations of onlyone or a few animal species. Therefore, the use of ME20.4-saporin conjugates both in primates [16,17] and insheep [19] suggests that Cy3-ME 20.4 might be successfullyemployed also in primates and ungulates, and become auseful cholinergic marker to determine morphological,electrophysiological and pharmacological parameters ofcholinergic neurons under physiological or pathologicalconditions.REFERENCES1. Záborszky L, Pang K, Somogyi J et al. Ann NY Acad Sci 77, 339–367(1999).2. Springer JE, Koh S, Tayrien MW and Loy R. J Neurosci Res 17, 111–118(1987).3. Tremere L, Brückner G, Brauer K et al. Brain Res 797, 351–356 (1998).4. Kordower JH, Bartus RT, Bothwell M et al. J Comp Neurol 277, 465–486(1988).5. Hefti F, Hartikka J, Salvaterra P et al. Neurosci Lett 69, 275–281 (1986).6. Wiley RG. Trends Neurosci 15, 285–290 (1992).7. Härtig W, Seeger J, Naumann T et al. Brain Res 808, 155–165 (1998).8. Kacza J, Grosche J, Seeger J et al. Brain Res 867, 232–238 (2000).9. Ha DH, Robertson RT and Weiss JH. 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Schnell SA, Staines WA and Wessendorf MW. J Histochem Cytochem 47,719–730 (1999).22. Jakab RL and Leranth C. Septum. In: Paxinos G, ed. The Rat NervousSystem. London: Academic Press; 1995, pp. 405–442.23. Harkany T, Grosche J, Mulder J et al. Neuroscience 108, 611–627 (2001).24. Kasaian MT and Neet KE. J Biol Chem 263, 5083–5090 (1988).25. Brauer K, Seeger G, Härtig W et al. J Neurosci Res 54, 248–253 (1998).Acknowledgements:The excellent technical assistance ofMrs Ute Bauer,Mrs Gabriele Lindner andMrs Gudrun Lemm is gratefullyacknowledged.This study was supportedby the Deutsche Forschungsgemeinschaft (grant Ha 2211/2-1toW.H. and C.V.), theInterdisziplin	res Zentrum fˇr Klinische Forschung (IZKF) at Leipzig University (grant 01KS 9504 projects Z10 and C5 to J.G. andJ.S., respectively) and the Hungarian National Science Foundation (OTKA, F035254, toT.H.).139 8 Vol 13 No 11 7 August 2002NEUROREPORT W.HHRTIG ETAL.

In vivo labeling of rabbit cholinergic basal forebrain neurons with fluorochromated antibodies

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In vivo labeling of rabbit cholinergic basal forebrain neurons with fluorochromated antibodies

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