Neurobiology of Aging 26 (2005) 1061–1072

Aging and subcellular localization of m2 muscarinic autoreceptorin basalocortical neurons in vivo

Marion Decossasa,b, Evelyne Doudnikoffa, Bertrand Blocha, Veronique Bernarda,c,!a Centre National de la Recherche Scientifique, Unite Mixte de Recherche 5541, Laboratoire d’Histologie-Embryologie,

Universite Victor Segalen-Bordeaux 2, 146 rue Leo-Saignat, 33076 Bordeaux Cedex, Franceb Centre National de la Recherche Scientifique, Unite Mixte de Recherche 5162, Laboratoire de Genomique Fonctionnelle des Trypanosomatides,

ATIPE Interaction Mitochondrie-Cytosquelette chez les Trypanosomes, Universite Victor Segalen-Bordeaux 2,146 rue Leo-Saignat, 33076 Bordeaux Cedex, France

c Biologie de la Synapse Cholinergique, Universite Rene Descartes—Paris 5, 45 rue des Saints-Peres, 75270 Paris Cedex 06, France

Received 12 March 2004; received in revised form 7 September 2004; accepted 24 September 2004

Abstract

By using immunohistochemical approaches at the light and electron microscopic levels, we have shown that aging modifies the subcellulardistribution of the m2 muscarinic autoreceptor (m2R) differentially at somato-dendritic postsynaptic sites and at axonal presynaptic sites incholinergic basalocortical neurons, in vivo. In cholinergic perikarya and dendrites of the nucleus basalis magnocellularis (NBM), aging isassociated with a decrease of the density of m2R at the plasma membrane and in the cytoplasm, suggesting a decrease of the total numberof m2R in the somato-dendritic field. In contrast, the number of substance P receptors per somato-dendritic surface was not affected. In thefrontal cortex (FC), we have shown a decrease of cytoplasmic m2R density also leading to a decrease of the number of m2R per surface ofvaricosities but with no change of the density of m2R at the membrane. Our results suggest that the decrease of m2R in the somato-dendriticfield of the NBM, but not a modification of the number of presynaptic m2 autoreceptors at the plasma membrane in the FC, could contributeto the decrease of the efficacy of cholinergic transmission observed with aging in the rat.© 2004 Elsevier Inc. All rights reserved.

Keywords: Acetylcholine; Aging; Autoreceptor; Receptor localization; Immunohistochemistry

1. Introduction

Over the past three decades, an exponentially growingnumber of investigations have focused on the relationshipbetween cognitive deficits and alterations of basal forebraincholinergic neurons in aging [2]. Impairments in a varietyof behavioral and cognitive functions have been attributed todeteriorating forebrain cholinergic systems in aged humans[32,33] and rodents [13,14,21,25,31]. So far, numerous stud-ies have thus focused on the effects of aging on the cholinergicbasalocortical pathway integrity in normal aged humans andanimals. The prevalent hypothesis is that aging is associatedwith dysfunctions in the basal forebrain cortical cholinergic

! Corresponding author. Tel.: +33 1 42 86 20 68; fax: +33 1 42 86 33 99.E-mail address: veronique.bernard@univ-paris5.fr (V. Bernard).

projection system. This is examplified by the decrease of thenumber of cholinergic neurons in the basal forebrain of agedrats [10,17,43]. Notably by using unbiased stereological ap-proaches and by counting ChAT immunoreactives neuronsand total Nissl-stained neurons, a decrease of the total num-ber of neurons in the basal forebrain due in part to a decreaseof the cholinergic ones has been demonstrated [41]. In agedhumans (90 year-old), the total number of neurons in the nu-cleus basalis of Meynert seems also decreased as comparedto young subjects (16–29 year-old) [11].Cholinergic receptors have been shown to play an impor-

tant role in the regulation of cognitive processes since theselective blockade of both muscarinic [45,49] and nicotinic[15,30] receptors induces memory deficits in young subjects,in comparison to those observed in old subjects. However,the effects of aging on the abundance of cholinergic recep-

0197-4580/$ – see front matter © 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.neurobiolaging.2004.09.007

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tors are still unclear.Measures of muscarinic receptor densityand affinity in the cortex of aged rats have also lead to conflict-ing conclusions about the effect of aging on the integrity offorebrain cholinergic neurotransmission [1,5,29,42]. Thesediscrepancies may be due to the use of different non-selectiveligands for muscarinic receptors. The availability of specifictools, i.e. receptor subtype antibodies, offers new methodsfor the quantification and localization of muscarinic recep-tor specific molecular subtypes. We bring here for the firsttime data concerning the subcellular distribution of one spe-cific molecular type of muscarinic receptor, m2R, in one spe-cific cell type, basalocortical cholinergic neurons, in differ-ent neuronal compartment, including somato-dendritic andaxonal fields. We have analyzed at the subcellular level thelocalization of m2R all along the somatodendroaxonal treeof the basalocortical neurons; at the membrane and in thecytoplasm. Notably, the m2 muscarinic receptor subtype isof particular interest in aging because this is the presynapticmuscarinic autoreceptor that negatively regulates the releaseof ACh from cortical cholinergic terminals [50]. In this con-text, we have determined, in young (6 months) and aged (24months) rats, the subcellular distribution of m2R at postsy-naptic (cell bodies and dendrites in the nucleus basalis mag-nocellularis (NBM)) and presynaptic (terminals in the frontalcortex (FC)) sites, in cholinergic neurons of the basalocorticalCh4 pathway. In order to do this, we have used immunohis-tochemistry at light and electron microscopic levels with am2R subtype selective antibody. We have studied as a con-trol, the subcellular localization of the substance P receptor(SPR) which, is colocalized with the m2R at the membraneof neurons in the NBM.

2. Materials and methods

2.1. Experimental groups

The experiments were performed on two groups of ani-mals: young rats (6months) and aged rats (24months) bred inthe laboratory (maleSprague–Dawley).All experimentswereperformed in accordance with the policy of the French Agri-culture and Forestry Ministry (decree 87849, licence 01499),with the approval of the Centre National de la RechercheScientifique and in accordance with the policy on the use ofanimals in neuroscience research issued by the Society forNeuroscience.

2.2. Tissue preparation

The rats were deeply anesthetized with sodium chloral hy-drate (150mg/kg) and at least four animals per group wereperfused transcardially with a mixture of 2% paraformalde-hyde and 0.2% glutaraldehyde [4]. Fifty micrometer-thicksections from NBM and FC were cut on a vibrating mi-crotome, collected in PBS, cryoprotected, freeze-thawed andstored in PBS with 0.03% sodium azide until use.

2.3. Immunohistochemistry

The m2R was detected by immunohistochemistry usinga monoclonal antibody raised in rat against a fusion pro-tein including a part of the third intracytoplasmic loop ofthe receptor (Chemicon, Temecula, CA). The specificity ofthe m2R antibody has previously been described in detail[3,22]. The m2R was analyzed specifically in cholinergicperikarya and dendrites in the nucleus basalis magnocellu-laris and in cholinergic varicosities in frontal cortex (FC)by combining immunodetection of m2R and choline acetyl-transferase (ChAT) or m2R and vesicular ACh transporter(VAChT), respectively, as previously described [12]. ChATand VAChT were detected using polyclonal antibodies bothraised in rabbit (Chemicon, Temecula, CA and a gift of Dr.R. Edwards, University of California at San Francisco, USA,respectively). The SPR was detected using a polyclonal anti-body raised in rabbit against a fusion protein that includes theC terminal domain of the receptor (a gift of Dr. R. Shigemoto,University of Kyoto, Japan) [40].

2.3.1. Immunofluorescence experimentsIn young rats, the m2R distribution in the cholinergic neu-

rons of the NBM was analyzed at light microscopic (LM)level by combining the simultaneous detection of m2R andChAT. The m2R and SPR relative distributions were also an-alyzed by combining the simultaneous detection of m2R andSPR by immunofluorescence.For the simultaneous detection of m2R and ChAT or m2R

and SPR, after perfusion-fixation as described above, sec-tions were incubated in 4% normal goat serum (NGS) for30min and then in a mixture of m2R (1:500) and ChAT(1:500) or m2R (1:500) and SPR (1:10,000) antibodies sup-plemented with 1% NGS for 15 h at room temperature (RT).The sections were then incubated in a mixture of goat anti-ratIgGs coupled to cyanine 3 (CY3) (Jackson ImmunoResearch,West Grove, USA) and goat anti-rabbit IgGs coupled to fluo-resceine isothiocyanate (FITC) (Jackson ImmunoResearch).All fluorochrome-conjugated antibodies were used at a dilu-tion of 1:200 in PBS for 1 h at RT. After washing, the sectionsweremounted in Vectashieldmountingmedium (Vector Lab-oratories, Burlingame, CA) and examined in a fluorescencemicroscope (Zeiss).

2.3.2. Immunogold experimentsThe m2R was analyzed at the electron microscopic (EM)

level specifically in cholinergic neurons of the NBM or incholinergic varicosities of the FC by combining m2R andChAT orm2R andVAChT immunodetection, respectively, aspreviously described [12]. The m2R was detected by the pre-embedding immunogold technique and ChAT andVAChT bythe pre-embedding immunoperoxidase using the peroxidaseanti-peroxidase (PAP) technique. Sections were incubated in4%NGS for 30min and then in amixture of m2R (1:500) andChAT (1:500) or m2R (1:5000) and VAChT (1:10,000) anti-bodies supplemented with 1%NGS for 15 h at RT. For the si-

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multaneous detection ofm2RandChAT in theNBM, sectionswere incubated in goat anti-rat IgGs coupled to biotin (Amer-sham, UK; 1:200) and goat anti-rabbit IgGs (Amersham;1:200) for 1 h 30. After washing, the sections were incubatedin PAP-rabbit (Sigma, France; 1:200) and in goat anti-biotinIgGs conjugated to gold particles (0.8 nm in diameter; Au-rion, The Netherlands; 1:100 in PBS/BSA-C) for 2 h. Thesections were then washed and post-fixed in 1% glutaralde-hyde for 10min. After washing in acetate buffer (0.1M, pH7), the signal of the gold immunoparticles was increased us-ing a silver enhancement kit (HQ silver; Nanoprobes, NY,USA) for 2min at RT in the dark. Finally, after washingin acetate buffer and in Tris buffer (TB; 0.05M, pH 7.6),the immunoreactive sites for ChAT were revealed by incuba-tion in 3,3-diaminobenzidine (DAB; Sigma, France; 0.05%in TB) in the presence of H2O2 (0.0048%). The reaction wasstopped by several washes in TB. For the simultaneous detec-tion of m2R and VAChT in cortex, m2R was detected usingthe tyramide signal amplification system (TSA;NewEnglandNuclear, Boston, USA). Sections of FC were incubated in amixture of goat anti-rat IgGs coupled to biotin (Amersham;1:200) and goat anti-rabbit IgGs (Amersham; 1:200) for 1 h30. The sections were incubated in streptavidin-HRP (NewEngland Nuclear; 1:100; 30min), then in biotinyl tyramide(New England Nuclear; 1:50; 7min), and finally in a mixtureof goat anti-biotin IgGs (0.8 nm in diameter; Aurion; TheNetherlands, 1:100) conjugated to gold particles and PAP-rabbit (DAKO, Denmark; 1:200) in PBS/BSA-C, for 2 h.The immunogold (m2R) and immunoperoxidase (VAChT)signals were revealed as described above. The sections werethen stored in acetate buffer 0.1M and processed for electronmicroscopy.The SPR was analysed at EM level using the immunogold

technique. Sections were incubated in 4% NGS for 30minand then in SPR (1:10,000) antibody supplemented with 1%NGS for 15 h atRT.Afterwashing, sectionswere incubated ingoat anti-rabbit IgGs conjugated to gold particles (0.8 nm indiameter; Aurion; The Netherlands, 1:100 in PBS/BSA-C).The immunogold signal was revealed as described above.

2.4. Preparation for electron microscopy

The sections were rinsed, post-fixed in 1% osmium tetrox-ide and dehydrated in ascending series of ethanol dilutions,that also included 70% ethanol containing 1% uranyl acetate.They were then treated with propylene oxide, impregnatedin resin overnight (Durcupan ACM; Fluka, Buchs, Switzer-land), mounted on glass slides and cured at 60 "C for 48 h.Areas of interest (the NBM and the layer 5 of the FC) werecut out from the sections and glued to blank cylinders ofresin. The observations were performed in layer 5 of the FCbecause this layer was shown to receive the most dense in-ervation from the cholinergic neurons of the NBM [38,39].Ultrathin sectionswere collected on pioloform-coated single-slot copper grids. The sections were stained with lead citrateand examined in Philips CM10 or Tecnai 20 EM.

2.5. Quantitative analysis

2.5.1. Immunofluorescence experimentsIn young rats, the percentage of ChAT immunopositive

cell bodies in the NBM that were also m2R immunoreac-tive was analyzed from immunofluorescence-treated sectionsdouble labelled for m2R and ChAT. Since some m2R im-munopositive neurons were not cholinergic, the percentageofm2R immunoreactive cell bodies in theNBM that were notChAT immunopositive was also quantified. In the NBM, allSPRpositive neuronswere seen to expressm2R. The percent-age of m2R positive neurons that were also immunopositivefor SPR was thus analysed. For these analysis, both hemi-spheres in two sections from four young and four old ratswere used.

2.5.2. Immunogold experimentsWe analyzed the variations of the subcellular localization

and the density of m2R and SPR in different subcellular el-ements. The distribution of m2R and SPR in the NBM andof m2R in the FC was analysed from immunogold-treatedsections at the electron microscopic level [12]. The analysisof the distribution of m2R in cholinergic neurons and of SPRin the NBM, was performed on negatives of micrographs ata final magnification of 2950 using the Metamorph software(Universal Imaging, Paris, France). After scanning the nega-tive (Magic Scan, version 3.1; Umax), the images were con-verted into positive pictures andmagnified to allow the identi-fication of the subcellular element showing immunoparticles.The measures were performed on four animals per group. Amean of 10 perikarya per animal (for m2R and SPR) and 40dendritic profiles (for m2R) were analyzed. In perikarya, theimmunoparticles were identified and counted in associationwith six subcellular compartments. The five compartmentsare the plasma membrane, endosome-like small vesicles, theGolgi apparatus, the endoplasmic reticulum and the outernuclear membrane. Some immunoparticles were classifiedas associated with a sixth unidentified compartment becausethey were associated either with no detectable organelles orwith an organelle that could not be identified as one of theprevious compartments. In dendrites, the distribution of theimmunoparticles was quantified in cholinergic dendritic pro-files associated with each quantified cholinergic neuron onthe same micrographs. All cholinergic dendrites on the pic-turewere used for the quantification. The percentage ofChATimmunopositive dendrites in the NBM that were also m2Rimmunoreactive was analyzed. A cholinergic dendrite wasconsidered as immunopositive when at least two particleswere detected. The immunoparticles were localized in as-sociation with three subcellular compartments: the plasmamembrane, endosome-like small vesicles and unidentifiedcompartments. The percentage of VAChT immunopositivevaricosities in the FC that were also m2R immunoreactivewas analyzed from immunogold-treated sections double la-belled for m2R and VAChT. All VAChT positive varicosi-ties from at least three ultrathin sections were counted under

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the EM and the percentage of the varicosities that were alsom2R positives was calculated. The analysis of the distribu-tion of m2Rwas performed using the Analysis software (SoftImaging System, GmbH, Germany) on a computer linked di-rectly to CCD camera on the electron microscope. A meanof 55 varicosities at a final magnification of 20,500# wasanalysed. The immunoparticles were localized in associationwith two subcellular compartments: the plasma membraneand the cytoplasm. Firstly, the results were expressed as theproportion of immunoparticles for m2R associated with thedifferent subcellular compartments in each neuron. For eachneuron, the number of immunoparticles associated with eachsubcellular compartment was counted and the proportion inrelation to the total number was calculated. Secondly, the

Fig. 1. Double detection of m2R (A, B, and C) and ChAT (A$, B$, and C$) inthe NBM of young rats using a double immunofluorescence method. (B–B$)A large-sized neuron immunolabelled for ChAT (B$) also expresses m2Rimmunoreactivity (B) mostly at the plasma membrane. A faint labeling form2R is seen in the cytoplasm of cell body and proximal dendrite. (C–C$)A large-sized neuron immunoreactive for m2R (C) at the plasma membraneof cell body and proximal dendrite is not labelled for ChAT (C$). (A–A$)A large-sized ChAT immunoreactive neuron (A$) is not immunoreactive form2R (A). Scale bar: 10!m.

results were also expressed in order to be able to comparethe variation of immunolabeling for m2R in each subcel-lular compartment between young and aged rats. For eachneuron, the number of immunoparticles associated with eachcompartment was counted in relation to membrane length(!m) for the plasma and nuclear membrane and to the sur-face of cytoplasm (!m2) for the endoplasmic reticulum, theendosome-like small vesicles and the unidentified compart-ment. For the Golgi apparatus, the values are expressed asthe number of immunoparticles per Golgi apparatus. We alsopresent a data called “total number of immunoparticles persurface of dendrite, soma or varicosity”, showing the totalnumber of immunoparticles quantified in the profile on themicrograph. Our quantification demonstrate variations of thequantity of immunoparticles per neuronal profile, which weassume that it is proportional to the absolute number of re-ceptors in these neuronal compartments. The values werecompared using non-parametric Mann–Whitney test.

3. Results

3.1. Phenotypic analysis of the m2R distribution incholinergic neurons of the NBM: light microscopicobservations

3.1.1. Young ratsThe double immunofluorescence experiments for m2R

and ChAT demonstrated that there were at least threesubpopulations of neurons in the NBM: neurons immuno-labelled for both m2R and ChAT (m2R+/ChAT+) and

Table 1Proportion of neurons immunolabelled for m2R and/or ChAT in the NBMof young and aged rats

m2R+ChAT ChAT

(A) ChAT immunoreactive neuronsYoung 87± 2% 13± 2%Aged 89± 1% 11± 1%

m2R+ChAT m2R

(B) m2R immunoreactive neuronsYoung 90± 1% 10± 1%Aged 88± 2% 12± 2%

Quantification of neurons immunoreactive for m2R and/or ChAT at lightmicroscopic level: analysis of double immunofluorescence experiments form2R (CY3) and ChAT (FITC). (A) Proportion of ChAT immunoreactivesneurons also labelled for m2R. For each animal, a mean of 49 ChAT im-munoreactive neurons was analysed. The percentages are expressed as themean±S.E.M. of the number of neurons double labelled for m2R and ChATor labelled only for ChAT, in relation to the total number of ChAT im-munoreactives neurons. (B) Proportion of m2R immunoreactive neuronsalso labelled for ChAT. For each animal, a mean of 48 m2R immunoreactiveneurons was analysed. The percentages are expressed as the mean±S.E.M.of the number of neurons double labelled for m2R and ChAT or labelledonly for m2R, in relation to the total number of m2R immunoreactive neu-rons. The statistical analysis (non-parametric Mann–Whitney test) shows nodifference in the proportion of neurons of each neuronal population betweenyoung and aged rats.

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neurons only labelled for m2R (m2R+/ChAT%) or forChAT (m2R%/ChAT+) (Fig. 1). The analysis at LM leveldemonstrated immunoreactivity for m2R in almost allneurons immunolabelled for ChAT in the NBM (87% ofChAT labelled neurons) (Table 1). Few neurons of the NBM(10% of m2R labelled neurons) showing immunoreactivityfor m2R but not for ChAT were also observed. A denseimmunoreactivity for m2R was associated with the plasmamembrane of perikarya and proximal dendrites (Fig. 1). Afaint staining was observed in the cytoplasm.

3.1.2. Aged ratsThe quantitative analysis at LM level of double m2R and

ChAT immunofluorescence experiments demonstrated thatthe percentage of neurons in each type of neuronal popu-lation (m2R+/ChAT+; m2R+/ChAT% and m2R%/ChAT+)was similar in young and old rats (Table 1). The propor-tion of neurons immunolabelled for ChAT and m2R wasunchanged in aged rats compared to controls (89 and 87%,respectively) and similarly, the proportion of neurons im-munolabelled for m2R but immunonegative for ChAT was

also unchanged in aged rats compared to controls (12 and10%, respectively).

3.2. Cellular and subcellular distribution of m2R in thecholinergic basalocortical neurons: electron microscopicobservations

3.2.1. Young rats3.2.1.1. Somato-dendritic compartment in theNBMof youngrats. Cellular and subcellular localization of m2RPerikarya: The ultrastructural studies of sections of NBM

double labelled form2R andChAT confirmed that a large partof m2R immunoparticles were associated with the plasmamembrane of cholinergic perikarya (43%) (Figs. 2 and 3A).In the cytoplasm, m2R immunoreactivity was present in sev-eral compartments including endoplasmic reticulum (15%),nuclear membrane (2.4%), Golgi apparatus (11%) and smallvesicles (1.6%). Twenty-seven percent of immunoparticleswere not associated with a recognizable organelle (Fig. 3A).Dendrites: Sixty-five percent of ChAT immunopositive

dendrites in the NBM expressed m2R (data not shown). Thequantitative analysis demonstrated that in cholinergic den-

Fig. 2. Subcellular distribution of m2R immunoreactivity in cholinergic perikarya (A and B) and dendrites (A$ and B$) of young and aged rats using the pre-embedding immunogold method with silver intensification. The cholinergic neurons are identified using the presence of immunoreactivity for ChAT, detectedby the immunoperoxidase method. The ChAT immunoreaction product is visible throughout the cytoplasm of the perikarya and dendrites. (A–A$) In youngrats, immunoparticles are mostly detected at the internal side of the plasma membrane (arrow heads) of cell bodies (A) and dendrites (A$). Few immunoparticlesare detected in the cytoplasm, some of them are associated with the endoplasmic reticulum (small arrow). (B–B$) In aged rats, the immunoparticles detected atthe internal side of the plasma membrane (arrow heads) of cell bodies (B) and dendrites (B$), and in the cytoplasm, in particular in the endoplasmic reticulum(small arrow), are less abundant than in young rats (A and A$). Scale bar: A–B: 1!m and A$–B$: 1!m.

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Fig. 3. Quantitative analysis of the subcellular distribution of m2R incholinergic perikarya of the NBM in young and aged rats using the pre-embedding immunogold method with silver intensification. (A) Proportionof immunoparticles for m2R associated with different subcellular neuronalcompartments in young rats. For each neuron, the number of immunopar-ticles associated with each subcellular compartment was counted and theproportion in relation to the total number was calculated. Data are expressedas percentage ±S.E.M. A large part of immunoparticles is associated withthe plasma membrane. In the cytoplasm, the immunoparticles are detectedin association with the endoplasmic reticulum (er) and the Golgi appa-ratus (Golgi). A small proportion of immunoparticles are associated withsmall vesicles (small ves.) and the nuclear membrane (n. mb). A part of im-munoparticles are not seen in association with any identified compartment(unident.). (B) Comparison of the localization of m2R immunoparticles indifferent subcellular compartments in cell bodies of cholinergic neuronsin the NBM of young and aged rats. For each neuron, the number of im-munoparticles associated with each compartment was counted in relationto the membrane length (!m) for the plasma membrane and the nuclearmembrane and to the surface of cytoplasm (!m2) for small vesicles, theendoplasmic reticulum and unidentified compartment. For the Golgi appa-ratus, the values are expressed as the number of immunoparticles per Golgiapparatus. Data are the result of counting four young and four aged rats in10 perikarya per animal. The results are expressed as the mean±S.E.M.in relation to an arbitrary unit (100) of the control values. In aged rats, thestatistical analysis (non-parametric Mann–Whitney test) shows a decreaseof the total number of immunoparticles associated with a decrease of thelabelling detected both at the plasma membrane and in the cytoplasm. In thecytoplasm of aged rats, the analysis demonstrates a decrease of the num-ber of immunoparticles associated with the endoplasmic reticulum and withnon-identified compartments. *p< 0.05; no symbol: not significant.

dritic shafts, immunoparticles were mostly associated withthe plasma membrane (78%) (Figs. 2 and 4A). In the cyto-plasm, m2R labelling was present in association with smallvesicles (2%) and with unidentified compartments (20%)(Fig. 4A).

Fig. 4. Quantitative analysis of the subcellular distribution ofm2R in cholin-ergic dendrites of the NBM in young and aged rats. (A) Proportion of im-munoparticles for m2R associated with different subcellular neuronal com-partments in young rats. For each cholinergic dendrite, the number of im-munoparticles associated with each subcellular compartment was countedand the proportion in relation to the total number was calculated. Results areexpress as percentage±S.E.M. The largest portion of immunoparticles is as-sociated with the plasma membrane. In the cytoplasm, the immunoparticlesare detected in association with small vesicles (small ves.) and with uniden-tified compartments (unident.). (B) Comparison of the localization of m2Rimmunoparticles in dendrites of cholinergic neurons in the NBM of youngand aged rats. For each dendrite, the number of immunoparticles associatedwith each compartmentwas counted in relation to themembrane length (!m)for the plasma membrane and to the surface of cytoplasm (!m2) for smallvesicles and unidentified compartment. Data are the result of countings infour young and four aged rats in 40 dendritic profiles per animal. The resultsare expressed as the mean±S.E.M. in relation to an arbitrary unit (100)of the control values. In aged rats, the statistical analysis (non-parametricMann–Whitney test) shows a decrease of the total number of immunopar-ticles associated with a decrease of the labelling at the plasma membrane.*p< 0.05; no symbol: not significant.

Cellular and subcellular localization of SPRThe analysis at the LM level in the NBM demonstrated

that all SPR positives neurons were also immunopositives form2R. This observation allowed the study of the subcellulardistribution of SPR as a control of the specifity of modifica-tions of ultrastructural m2R localization. A dense immunore-activity for m2R and SPR was associated with the plasma

M. Decossas et al. / Neurobiology of Aging 26 (2005) 1061–1072 1067

Fig. 5. Double detection of m2R (A) and SPR (B) in the NBM of youngrats using a double immunofluorescence method. A large-sized neuron isimmunolabelled for m2R (A) and for SPR (B), both prominently at theplasma membrane of cell body and proximal dendrite.

membrane of perikarya and proximal dendrites (Fig. 5).SPR immunoreactivity was present in 82% of m2R positivesneurons (Table 2). Moreover, in accordance with previousstudy [7], our results demonstrated that all SPR immunore-actives neuronswere also immunolabelled forChAT (data notshown). Ultrastructural studies of sections of NBM labelledfor SPR show that a large part of SPR immunoparticles wereassociatedwith the plasmamembrane (47%) (Figs. 6 and7A).In the cytoplasm, SPR immunoreactivity was present in sev-

Table 2Proportion of neurons immunolabelled for m2R and/or SPR in the NBM ofyoung and aged rats

m2R±SPR m2R

m2R immunoreactive neuronsYoung 82± 2% 18± 2%Aged 84± 1% 16± 1%

Quantification of neurons immunoreactive for m2R and/or SPR at lightmicroscopic level: analysis of double immunofluorescence experimentsfor m2R (CY3) and SPR (FITC). For each animal, a mean of 47 m2Rimmunoreactive neurons was analysed. Percentages are expressed as themean±S.E.M. of the number of neurons double labelled for m2R and SPRor labelled only for m2R, in relation to the total number of m2R immunore-active neurons. The statistical analysis (non-parametricMann–Whitney test)shows no difference in the percentage of neurons expressing m2R also la-belled for SPR, between young and aged rats.

eral compartments including endoplasmic reticulum (17%),nuclear membrane (2%), Golgi apparatus (7%) and smallvesicles (1%). Twenty-six percent of immunoparticles werenot associated with a recognizable organelle (Fig. 7A).

3.2.1.2. Axonal compartment in the FC of young rats. Thequantification of sections double labelled for m2R andVAChT at EM level demonstrated that 23% of cholinergicvaricosities (VAChT immunopositive) expressed m2R im-

Fig. 6. Subcellular distribution of SPR immunoreactivity in the NBM of young and aged rats using the pre-embedding immunogold method with silverintensification. In young (A) and aged (B) rats, immunoparticles are mostly detected at the internal side of the plasma membrane (arrow heads) of cell bodies(A and B). Few immunoparticles are detected in the cytoplasm, some of them are associated with the endoplasmic reticulum (small arrow). Scale bar: 1!m.

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Fig. 7. Quantitative analysis of the subcellular distribution of SPR inperikarya of the NBM in young and aged rats using the pre-embedding im-munogold method with silver intensification. (A) Proportion of immunopar-ticles for SPRassociatedwith different subcellular neuronal compartments inyoung rats. For each neuron, the number of immunoparticles associated witheach subcellular compartment was counted and the proportion in relation tothe total number was calculated. Data are expressed as percentage±S.E.M.A large part of immunoparticles is associated with the plasma membrane.In the cytoplasm, the immunoparticles are detected in association with theendoplasmic reticulum (er) and the Golgi apparatus (Golgi). A small pro-portion of immunoparticles are associated with the nuclear membrane (n.mb) and small vesicles (small ves.). A part of immunoparticles are not seenin association with any identified compartment (unident.). (B) Comparisonof the localization of SPR immunoparticles in different subcellular compart-ments in cell bodies of the NBM in young and aged rats. For each neuron, thenumber of immunoparticles associated with each compartment was countedin relation to the membrane length (!m) for the plasma membrane and thenuclear membrane and to the surface of cytoplasm (!m2) for the endoplas-mic reticulum, small vesicles, and unidentified compartment. For the Golgiapparatus, the values are expressed as the number of immunoparticles perGolgi apparatus. Data are the result of counting four young and four aged ratsin ten perikarya per animal. The results are expressed as the mean±S.E.M.in relation to an arbitrary unit (100) of the control values. In aged rats, thestatistical analysis (non-parametric Mann–Whitney test) shows a decreaseof the number of immunoparticles associated with small vesicles. No othermodification of the SPR subcellular distribution was noted in aged rats.*p< 0.05; no symbol: not significant.

munoreactivity (data not shown). The analysis at the ul-trastructural level demonstrated that 83% of immunopar-ticles for m2R were associated with the plasma mem-brane of VAChT immunolabelled cholinergic terminals(Figs. 8 and 9A). Seventeen percent of immunoparticles were

Fig. 8. Subcellular distribution of m2R immunoreactivity in cholinergicvaricosities of the frontal cortex in young and aged rats using the pre-embedding immunogold method with silver intensification. The cholinergicvaricosities are identifiedusing the presenceof immunoreactivity forVAChT,detected by the immunoperoxidase method. The VAChT immunoreactionproduct is visible throughout the cytoplasm of the varicosities. In young (A)and aged (B) rats, immunoparticles are mostly detected at the internal sideof the plasma membrane of cholinergic varicosities. Scale bar: 0.25!m.

detected in the cytoplasmwhere theywere not associatedwithidentified compartments (Fig. 9A).

3.2.2. Aged rats3.2.2.1. Somato-dendritic compartment in the NBM of oldrats. Cellular and subcellular localization of m2RPerikaya: The quantitative analysis at EM level of sec-

tions of NBM double labelled for m2R and ChAT demon-strated a decrease of the total number of immunoparticlesper neuronal surface in old rats (%52%) (Fig. 3B). Theanalysis of the labelling associated with the different sub-cellular neuronal compartments demonstrated that the den-sity of m2R decreased at the plasma membrane in old rats(%51%) (Figs. 2 and 3B). In the cytoplasm of cholinergicneurons, a decrease of the number of immunoparticles form2R associated with endoplasmic reticulum (%52%) andwith the unidentified compartment (%44%) was also ob-served (Fig. 3B).Dendrites: Our quantification data demonstrated a signif-

icant decrease of the number of ChAT positive dendrites alsoimmunoreactive for m2R in old rats (%24%, data not shown).In cholinergic dendritic shafts, the ultrastructural analysisdemonstrated a decrease (%37%) of the total number of im-munoparticles per dendritic surface in old rats (Fig. 4B).Moreover, the analysis of the labelling associated with thedifferent subcellular dendritic compartments demonstrateda significant decrease of the density of immunoparticles lo-cated at the plasma membrane in old rats (%30%) (Figs. 2B$

and 4B).Cellular and subcellular localization of SPRIn accordance with our results obtained in young rats, the

quantitative analysis at LM level of double immunofluores-cence experiments for m2R and SPR demonstrated that allSPR positives neurons also express m2R. In aged rats, SPRis present in 84% of m2R positives neurons (Table 2).The quantitative analysis at EM level of sections of NBM

immunolabelled for SPR showed no modification of the den-sity of SPR located at the plasma membrane in old rats

M. Decossas et al. / Neurobiology of Aging 26 (2005) 1061–1072 1069

Fig. 9. Quantitative analysis of the subcellular distribution ofm2R in cholin-ergic varicosities of the frontal cortex in young and aged rats. (A) Proportionof immunoparticles for m2R at the plasma membrane or in the cytoplasm ofcholinergic varicosities in young rats. The number of immunoparticles form2R in cholinergic varicosities, localized at the plasma membrane or in thecytoplasm was counted and the proportion in relation to the total numberwas calculated. The largest portion of immunoparticles is associated withthe plasma membrane. In the cytoplasm, the immunoparticles are detectedin association with unidentified compartments (unident.). (B) Comparisonof the localization of m2R immunoparticles in cholinergic varicosities ofthe FC, in young and aged rats. For each varicosity, the number of im-munoparticles associated with each compartment was counted in relation tothe membrane length (!m) for the plasma membrane and to the surface ofcytoplasm (!m2) for the unidentified compartment. Data are the result ofcountings in four young and four aged rats in 55 varicosities per animal.The results are expressed as the mean±S.E.M. in relation to an arbitraryunit (100) of the control values. In aged rats, the statistical analysis (non-parametric Mann–Whitney test) shows a decrease of the total number ofimmunoparticles associated with a decrease of the density of immunoparti-cles associated with non-identified compartments. *p< 0.05; no symbol: notsignificant.

(Figs. 6 and 7B). However, the analysis demonstrated a de-crease of the number of immunoparticles for SPR detectedin association with small vesicles (%61%) (Fig. 7B).

3.2.2.2. Axonal compartment in the FC of old rats. Thequantitative analysis at the ultrastructural level, demonstrateda significant decrease of the density of cytoplasmic im-munoparticles (%55%) leading to a decrease of the totalnumber of immunoparticles per terminal surface (%20%)

(Figs. 8 and 9B). However, no modification of the densityof immunoparticles located at the plasma membrane was ob-served (Figs. 8 and 9B). The number of VAChT positive vari-cosities also immunoreactive form2Rwas notmodified in oldrats (22%, data not shown).

4. Discussion

We report here the first description of the effect of ag-ing on the subcellular localization of the m2 autoreceptor inall parts of the basalocortical cholinergic neurons. We havedemonstrated that the subcellular distribution and membraneabundance ofm2R ismodifiedwith aging and that thesemod-ifications vary with neuronal compartments: soma/dendritesin the NBM or terminals in the FC. (1) In control animals, inaccordance with previous data obtained in mice [12], m2Ris prominently located at the plasma membrane at postsy-naptic sites in the NBM and at presynaptic sites in corticalvaricosities. (2) In the somato-dendritic compartment in theNBM of aged rats, the percentage of cholinergic dendritesexpressing m2R decreases. In parallel, a decrease of the totaldensity of m2R immunoparticles associated with a depletionof cell surface receptors were observed in cholinergic cellbodies and dendrites of the NBM. This was accompanied bya decrease of cytoplasmic m2R immunoreactivity due to adecrease of the density of receptors associated with the en-doplasmic reticulum and the unidentified compartments. Incontrast, in aged rats, few changes were observed for SPR inneurons of the NBM. In the FC, the percentage of choliner-gic varicosities expressing m2R was unchanged with agingand the density of plasma membrane m2R was not modified.However, the cytoplasmic number of receptorswas decreasedin cortical cholinergic varicosities of aged rats leading to adecrease of the total number of m2R immunoreactive sites.

4.1. Effect of aging on the cellular and subcellulardistribution of postsynaptic m2R in the NBM

In the NBM of aged rats, the decrease of the total num-ber of m2R in cholinergic cell bodies and dendrites and thedecrease of the number of cholinergic dendrites expressingm2Rdemonstrate thatm2R is downregulatedwith aging.Thisdownregulation is associated with a decrease of cell surfacereceptors in cholinergic neurons of the NBM. Very few datahave focused on the status of m2R in the NBM of normalaged rats. Most studies have concerned the target brain ar-eas of cholinergic neurons including cortex, hippocampus oramygdala where the cholinergic deficits correlates with cog-nitive decline in pathological aging, i.e. in Alzheimer’s dis-ease [18,19,44]. However, our results are in accordance withthe few existent data. Autoradiographic studies have reporteda decrease ofM2muscarinic receptors in the cholinergic basalforebrain of aged rats [5,29]. This M2 pharmacological typecorrespond to m2R and m4R. It has been demonstrated thatthe m4R is not expressed or at very low level compared to

1070 M. Decossas et al. / Neurobiology of Aging 26 (2005) 1061–1072

m2R, in the cholinergic basal forebrain [23,47]. Thus, this de-crease inM2binding sites certainly reflects a decrease ofm2Rbinding sites in these structures. Our study demonstrated thatthis decrease occurred specifically in cholinergic neurons.Our results bring the first demonstration of the subcel-

lular events that may contribute to downregulation of m2R:a decrease of the density of m2R at the plasma membraneand a decrease of the number of m2R associated with theendoplasmic reticulum. This decrease of the density of m2Rassociated with the endoplasmic reticulum may be explainedby alterations of the m2R gene expression. In accordancewith this hypothesis, a decrease of the level of m2R mRNAin old rats has been demonstrated in different brain structuresincluding cholinergic basal forebrain nuclei [29]. Thus, a de-crease of m2R synthesis may lead to a decrease of the totalnumber of m2R in neurons, and notably to a decrease of cellsurface receptors. Alterations in the targeting of m2R at theplasma membrane may also contribute to the decrease of theexpression ofm2R at the cholinergic cell surface in theNBM.However, the modifications of the subcellular distribution ofm2R do not seem to be due to a general decrease of the activ-ities of synthesis andmembrane targeting of proteins, includ-ing other neurotransmitter receptors, in these neurons sincethe density of SPR in the endoplasmic reticulum and at theplasma membrane are unaltered in aged rats. This suggeststhat aging specifically affects the subcellular distribution ofcholinergic m2 receptors.Alterations in different processes regulating m2R synthe-

sis, which could involve ACh or other messengers may alsocontribute to the decrease of the abundance of m2R at theplasma membrane. In this context, alterations of the sup-ply in neurotrophic factors in aging, which is known to reg-ulate gene expression [28], including muscarinic receptors[8,16,36] may contribute to the regulation of the subcellu-lar distribution of m2R. Notably, exposure to NGF of telen-cephalic neurons modulates gene expression and abundanceof binding sites of the different muscarinic receptors [16].Trophic factors are also known to regulate muscarinic re-ceptors gene expression in neurons of the superior cervicalganglia [24].

4.2. Effect of aging on the cellular and subcellulardistribution of presynaptic m2R in the FC

We have demonstrated here that aging induces a decreaseof the density of m2R in cholinergic varicosities of the FC,which is due to a decrease of cytoplasmic receptors with nomodification of the abundance of m2R at the plasma mem-brane. This is the first demonstration that the compartmental-ization of the m2R in cholinergic cortical terminals is modi-fied with aging. An hypothesis to explain these results, maybe that in normal conditions, a pool of intracytoplasmic recep-tors could be recruited for membrane targeting. With aging,this pool of receptors may be progressively downregulatedfavouring the targeting of receptors to the membrane. Alter-ations in axonal transport of m2R may also contribute to this

decrease of m2R in cholinergic terminals. In this context,although not specifically shown for cholinergic neurons, al-terations in cortical axonal transport have been demonstratedin aged people suffering from Alzheimer’s disease [9].Ourwork bring for the first time specific information about

the effects of aging on the m2R as a presynaptic autorecep-tor. In fact, most autoradiographic studies have been devotedto the status of M2 binding sites in the cortex of aged ratsand thus of m2R since m4R is not expressed or is expressedat very low levels in the rat cortex [23,48]. However, au-toradiography takes in account pre- and postsynaptic recep-tors and it is now well established that some noncholiner-gic neurons expressed m2R as a postsynaptic receptor in thecortex of the rat. In addition, this technique does not dis-criminate between auto- and hetero-receptors since m2R isalso expressed by non-cholinergic terminals in the frontalcortex [23,26,37]. In this context, our system of analysis ap-peared the most suitable to specifically study the status ofm2R in cholinergic terminals and thus to bring informationconcerning m2R as the presynaptic autoreceptor in the FC.Moreover, autoradiographic studies of muscarinic receptorsin aged rats, have lead to conflicting results sinceM2 bindinghas been reported both increased [35] or decreased [1,5,29]in the cortex of aged rats. These discrepancies could be ex-plained by differences in the specificity of the ligands or inbinding methods. In aged humans, a decrease of the bindingof M2 receptors in cortical synaptosomes, that are locatedat presynaptic sites, has been reported [32]. Alternatively, anincrease of the binding of M2 in aging has been reported inhumans in vivo [34]. This discrepancy in the modificationsof M2 binding may be due to species differences. It mayalso be attributed to postsynaptic receptors since m2R hasbeen demonstrated to be expressed by some cortical neurons.The pre- and postsynaptic m2R may be submitted to oppo-site regulations, as we demonstrate for m2R in the presentwork. Since the ligand used by Podruchny et al. (2003) isspecific for m2R [20] and since we show here a decrease ofm2R immunoreactivity in cholinergic terminals, the increaseof M2R binding may be due to an increase of postsynap-tic m2R. Moreover, the decrease of ACh during aging maylead to a decrease of the number of receptors occupied bythe native ligand and thus an increase of free sites for theradioactive ligand without change in the abundance of m2Rat the plasma membrane as we describe in this study. Al-ternatively, the affinity of receptors but not the total numberof receptors may have been modified. Recent studies, us-ing knock-out mice, have indicated that auto-inhibition ofACh release is mediated by m2R in the cortex [50]. Corti-cal potassium-induced ACh release is decreased in aged rats.However, blockade or activation of muscarinic receptors byatropine [27] or oxotremorine [6], increases or decreases, re-spectively, ACh release in the same extend in aged and youngrats. This suggests that muscarinic presynaptic regulation ofACh is unalteredwith aging. In this context, our results show-ing no modification of the membrane abundance of m2R inaged rats, together with these previous data, suggest that m2R

M. Decossas et al. / Neurobiology of Aging 26 (2005) 1061–1072 1071

functions as presynaptic autoreceptor are unaltered with ag-ing.A decrease of the number of cholinergic terminals associ-

ated with a shrinkage of persistent ones have been reported inthe cortex of aged rats [46]. However, even if the number ofcholinergic varicosities is decreased, we have demonstratedthat the proportion of these terminals expressing m2R is un-changed with aging. Thus, aging might specifically affectcholinergic terminals that do not express m2R which couldexplain the difference of results concerningACh releasewhendifferent stimuli are used (local depolarisation or blockade ofmuscarinic presynaptic receptors).

5. Conclusion

Our results suggest that cholinergic dysfunctions observedwith aging are associated with change in expression and lo-calization of m2R. The decrease of the density of m2R atthe plasma membrane in the NBM may alter neuronal ac-tivity triggered by the stimulation of these receptors. Thus,modifications of the cholinergic neuronal reactivity inducedby the decrease of the number of membrane receptors maycontribute to the general decrease of the cholinergic basalo-cortical transmission with aging.We have observed different effects of aging on the density

of m2R at the plasma membrane in somato-dendritic and ax-onal compartments. This suggests that the mechanisms reg-ulating the membrane targeting of m2R differs according toits localization at the plasma membrane in the different partsof the neuron and that these mechanisms are differentiallyaltered with aging.Our anatomical results, showing that the availability of

m2R at the membrane of cholinergic terminals in the frontalcortex are not altered with aging, are consistent with the pre-vious biochemical findings demonstrating that the inhibitoryeffect of m2R on ACh release is still present in the cortex ofaged rats [6,27].

Acknowledgments

The authors thank Dr. R. Edwards (University of Califor-nia at San Francisco, CA) and Dr. R. Shigemoto (Universityof Kyoto, Japan) for supplying us with the VAChT and sub-stance P receptor antibodies, respectively. We also thank Dr.F. Dellu for her helpful discussions, Dr. Derrick Robinson forreading and commenting this manuscript, M. Leguet and S.Weber for animal care and the Electron Microscopy Centreof the University Victor Segalen-Bordeaux2.

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