m2 muscarinic acetylcholine receptor-immunoreactive neurons are not reduced within the nucleus...

m2 Muscarinic AcetylcholineReceptor-Immunoreactive Neurons AreNot Reduced Within the Nucleus Basalis

in Alzheimer’s Disease: Relationship WithCholinergic and Galaninergic Perikarya

E.J. MUFSON,1,2* S. JAFFAR,1,2 AND A.I. LEVEY3

1Department of Neurological Sciences, Rush Presbyterian-St. Luke’s Medical Center,Chicago, Illinois 60612

2Rush Alzheimer’s Disease Center, Rush Presbyterian-St. Luke’s Medical Center,Chicago, Illinois 60612

3Department of Neurology, Emory University, Atlanta, Georgia 30322

ABSTRACTThe distribution of profiles containing the m2-muscarinic acetylcholine receptor within

the aged human basal forebrain and its relationship to cholinergic and noncholinerigcneurons and alterations in Alzheimer’s disease (AD) was investigated by using an m2-specificmonoclonal antibody (Levey et al. [1995] J. Comp. Neurol. 351:339–356). Immunocytochemis-try revealed that the m2 receptor protein is expressed primarily in noncholinergic multipolarneurons (mean 6 S.E.M.; 355.7 6 15.4 µm2) located primarily outside the cholinergic nucleusbasalis subfields. Dual-stained sections from the septal/diagonal band complex revealed thatm2 was detected in only 14% of choline acetyltransferase-labeled neurons, whereas only 13%of m2-stained neurons colocalized choline acetyltransferase. Within the nucleus basalis,choline acetyltransferase was found in 35% of the m2-labeled neurons, whereas only 6% ofcholine acetyltransferase-stained perikarya were dual labeled for m2. Although we did notvisualize colocalization of m21/choline acetyltransferase-immunonegative neurons and theinhibitory neuropeptide galanin, galaninergic fibers coursed in close apposition to m21 cells.Cell counts demonstrated that 90% of choline acetyltransferase-labeled striatal neuronsexpressed the m2 receptor. Despite the extensive reduction in cholinergic basal forebrainneurons, cell counts of the relative number of m2-immunoreactive neurons within the nucleusbasalis complex from aged controls and AD patients revealed that the m2 neurons are spared.Finally, our findings suggest that most m2 receptors in the cholinergic basal forebrain arelocated on noncholinergic structures; therefore, they are not the major source of m2 receptorsin the cortex. Thus, the reduced levels of the m2 receptor seen in AD cortex probably reflectchanges in other neuronal populations. J. Comp. Neurol. 392:313–329, 1998.r 1998 Wiley-Liss, Inc.

Indexing terms: aging; basal forebrain; choline acetyltransferase; disease; galanin; human;

immunohistochemistry

Cholinergic transmission in the neocortex and hippocam-pus plays a crucial role in higher cognitive processes, suchas learning and memory (see, e.g., Bartus et al., 1982;Fibiger, 1991). These functions, for the most part, aremediated by muscarinic acetylcholine (ACh) receptors(mAChRs). Several lines of evidence have implicated im-paired muscarinic cholinergic systems as contributing tothe cognitive impairment seen in Alzheimer’s disease(AD), including 1) the consistent depletion of choline

Grant sponsor: NIH; Grant numbers: AG10688, AG10161, AG11482,AG09466, AG10130, AG14449, NS30454, and NS31937.

*Correspondence to: Elliott J. Mufson, Ph.D., Rush Presbyterian-St.Luke’s Medical Center, Tech 200, 2242 W. Harrison St., Suite 200, Chicago,IL 60612. E-mail: [email protected]

Received 21 July 1997; Revised 8 October 1997; Accepted 15 October1997.

THE JOURNAL OF COMPARATIVE NEUROLOGY 392:313–329 (1998)

r 1998 WILEY-LISS, INC.

acetyltransferase (ChAT) in neocortex and hippocampus inboth early- and late-onset forms of the disease (Perry,1978; Coyle et al., 1983; Etienne et al., 1986); 2) theimpaired cognition in animals and humans as a result ofcholinergic basal forebrain (CBF) lesions and pharmacologi-cal blockade of mAChR (Drachman and Leavitt, 1974;Damasio et al., 1985; Dunnett, 1985; Nilsson et al., 1992);3) the reduction of CBF cortical projection neurons in ADpatients (Whitehouse et al., 1981; Arendt et al., 1983;Mufson et al., 1989b); 4) the correlation of ChAT levels(Perry, 1978; Coyle et al., 1983) and numbers of basalforebrain neurons (Whitehouse et al., 1981; Doucette et al.,1986; Lehericy et al., 1993) with the severity of dementia(Wilcock et al., 1982); and 5) the improvement of cognitionand behavior in AD patients by selective muscarinicagonists (Bodick et al., 1997). Thus, although it is clearthat many brain regions and types of neurons degeneratein AD patients (Biere et al., 1995), hypofunction of cholin-ergic neurotransmission mediated by mAChRs appears toplay an important role in AD symptomatology.

Recently, several muscarinic receptor subtypes havebeen identified by differential affinities for antagonists,and it is now established that five distinct genes, m1–m5,encode highly related muscarinic receptor subtypes (Bon-ner et al., 1987; Hulme et al., 1990; Vilaro et al., 1994).Most studies of muscarinic receptor alteration with ADhave concentrated on changes in the pharmacologicallydefined binding sites in the cortex of aged normal controlsand in AD postmortem brain (for review, see Levey, 1996).These findings have led to the suggestion that ‘‘postsynap-tic’’ receptors are largely unchanged in number in ADbrains (Mash et al., 1985; Levey et al., 1995) but may notbe functional (Flynn et al., 1991). There has been lessconsensus regarding ‘‘presynaptic’’ receptors, but somestudies have found that these are reduced in AD (Mash etal., 1985; Flynn et al., 1995). The M2 class of receptors(defined pharmacologically) has received much attentionas a presynaptic receptor due to its relationship withcholinergic neurons and its reduction in AD (Mash andPotter, 1985; Flynn et al., 1995). It is often assumed thatm2 is the gene product that functions as a presynapticautoreceptor to inhibit ACh release (Mash et al., 1985;Mash and Potter, 1986; Pohorecki et al., 1988; Levey et al.,1991) and, like the ‘‘M2’’ radioligand binding sites, m2-immunoreactive protein is reduced in AD brain (Flynn etal., 1995). However, only a subset of cholinergic neuronscoexpress the m2 protein within the rodent basal forebrain(Levey et al., 1995), suggesting that these neurons are notthe only source of the m2 receptor protein seen in thehippocampus, cortex, and other terminal zones. In supportof this concept, lesions of the rodent septohippocampal andmonkey nucleus basalis cortical projection systems resultin little, if any, decrease in m2 receptor protein in thehippocampus and cortex, respectively (Wall et al., 1994;Levey et al., 1995; Mrzljak et al., 1997). Despite theseintriguing findings in animals, very little is yet knownabout the expression of m2 in the human basal forebrainand possible alterations in AD. Therefore, the purpose ofthe present study was to evaluate critical aspects of m2localization necessary for understanding the role of thisreceptor within the human basal forebrain, including theprecise cellular distribution of the receptor protein, itsrelationship to cholinergic and noncholinerigc neurons,and alterations related to AD. By using a recently devel-oped m2-specific monoclonal antibody (Levey et al., 1995),

with immunocytochemistry, we found that the m2 receptorprotein is expressed primarily in noncholinergic basalforebrain neurons in the normal aged human brain and,surprisingly, that these neurons are spared in AD brains.

MATERIALS AND METHODS

Tissue preparation

Brains from five patients (mean age, 84.8 years) diag-nosed with a clinical history of AD, which was confirmedupon postmortem neuropathological analysis, and fiveaged normal control cases (mean age, 68.7 years) who wereclinically nondemented were examined following autopsy(see Table 1). Following removal from the calvarium, eachbrain was cut on a brain slice apparatus into 1-cm-thickslabs, immersion fixed in 4% paraformaldehyde (FisherScientific, Itasca, IL) in 0.1 M phosphate buffer for 24–48hours at 4°C, and cryoprotected in 10% glycerol plus 2%dimethyl sulfoxide (DMSO) in 0.1 M phosphate buffer at4°C for 2 days, followed by a solution of 20% glycerol plus2% DMSO (Rosene et al., 1986). Brain slabs containing thesubfields of the nucleus basalis were cut into several seriesof adjacent 40-µm-thick sections on a freezing, slidingmicrotome and were stored in a cryoprotectant (Mufson etal., 1989a, 1993) until processed. Because asymmetriesbetween hemispheres within the basal forebrain do notoccur in humans (Doucette and Ball, 1987), the datagenerated from one hemisphere should reflect both sides ofthe brain. Sections containing the medial septum/diagonalband complex (Ch1/Ch2) were not always available foreach case (n 5 3). Therefore, in these cases, only tissuefrom the nucleus basalis (Ch4) was examined. The lefthemisphere was paraffin embedded for neuropathologicalevaluation. Sections from each human brain containingthe amygdala, hippocampal complex, and temporal cortexwere examined for neuritic plaques and neurofibrillarytangles with thioflavin-S and Bielschowsky silver stains,according to previously described protocols (Mufson et al.,1989b, 1993). Each of the neo- and limbic cortical areasexamined in AD brains exhibited numerous neuriticplaques, meeting the age-adjusted Kachaturian (1985)criteria for this disease. Neuropathologic evaluation alsorevealed that many basal forebrain neurons exhibitedglobose-shaped neurofibrillary tangles, as previously re-ported (Mufson et al., 1989b). Control cases were nonde-mented clinically and failed to fulfill the age-adjustedneuropathological National Institute for Aging/Alzheimer’s

TABLE 1. Case Demographics1

CaseAge

(years) SexBrain

weight (g)PMI

(hours)ApoE

genotype

Controls1. 72 M 1,050 7.0 2/32. 59 M 1,250 5.5 N/A3. 85 M 1,260 17.0 3/34. 59 M 1,430 19.0 3/35. 68 M N/A N/A N/AMean 68.6 1,247.5 12.1S.E.M. 4.8 77.7 3.4

Alzheimer’s6. 94 M 920 3.0 3/37. 83 F 1,140 8.0 3/48. 82 M 1,285 6.0 3/49. 83 F 990 5.5 3/4

10. 82 M 1,270 5.5 3/4Mean 84.8 1,121 5.6S.E.M. 2.3 73.1 0.7

1PMI, postmortem interval; ApoE, apolipoprotein E; N/A, not available.

314 E.J. MUFSON ET AL.

Fig. 1. Low-magnification photomicrographs of adjacent stainedsections comparing the distribution of cholinergic (A–C) and m2 (D–F)immunoreactivity within the aged normal human basal forebrain.Cholinergic profiles were visualized by using an antibody against thelow-affinity p75 neurotrophin receptor (p75NTR; see text for details).There was a virtual complete overlap between the distribution ofp75NTR and m2 neuropil staining within the diagonal band (Ch2) and

the anteromedial (Ch4am) and anterolateral (Ch4al) subfields of thebasal forebrain. In contrast, only m2 immunoreactivity was seen inthe caudate (CD) and putamen (PT) as well as the bed nucleus of thestria terminals (BNST). AC, anterior commissure; fx, fornix; GP,globus pallidus; GPe, globus pallidus external; GPi, globus pallidusinternal; ic, internal capsule; LV, lateral ventricle. Scale bar 5 3 mm.

m2 RECEPTORS IN ALZHEIMER’S DISEASE 315

Figure 2


Disease and Related Disorders Association (NIA/ADRDA)criteria for AD.

Antisera

Immunohistochemistry was performed by using a ratmonoclonal antibody specific for the m2 receptor. Thepreparation and characterization of the antibody has beendescribed previously in detail (Levey et al., 1995). ChATimmunohistochemistry was performed by using a poly-clonal rabbit antiserum against ChAT from human pla-centa (German et al., 1985; Bruce et al., 1985; Mufson etal., 1988a,b, 1989a). Immunohistochemistry for the visual-ization of the low-affinity p75 neurotrophin receptor(p75NTR) was performed by using a monoclonal antibodyraised against human p75NTR (Schatteman et al., 1988;Mufson et al., 1989a). Galanin (GAL) immunohistochemis-try was performed with a rabbit anti-GAL polyclonalantisera (Peninsula Laboratories, Belmont, CA) directedagainst a synthetic porcine GAL peptide (Kordower andMufson, 1990; Kordower et al., 1992; Benzing et al., 1993;Mufson et al., 1993).

Immunohistochemistry

Adjacent series of sections were processed for the visual-ization of m2 and other markers in the basal forebrain.Each antibody (m2 diluted 1:400, p75NTR diluted 1:60,000,ChAT diluted 1:1,800, and GAL diluted 1:15,000) wasapplied for 24 hours at room temperature. Immunohisto-chemistry was performed according to previously reportedprotocols (Mufson et al., 1989a,b, 1993; Benzing et al.,1993; Levey et al., 1995). Briefly, following several rinses,sections were incubated in biotinylated goat anti-rat immu-noglobulin (IgG; m2), horse anti-mouse IgG (p75NTR), orbiotinylated goat anti-rabbit IgG (ChAT and GAL) second-ary antibody diluted 1:200 (Vector Laboratories, Burlin-game, CA) for 60 minutes, followed by incubation in aavidin-biotin complex for 60 minutes at 1:500 dilution(ABC Elite Kit; Vector Laboratories). The reaction wasfinalized with a modification of the Hancock (1982) method.Sections were washed six times for 10 minutes each in a0.2 M sodium acetate-1.0 M imidazole buffer, pH 7.4. Thechromagen solution contained 2.5% nickel II sulfate (FisherScientific), 0.05% 3838-diaminobenzidine (DAB), and 0.005%

H202, pH 7.2. The reaction was terminated with washes inacetate-imidazole buffer.

Immunohistochemical double-labelprocedures

Colocalization of m2 and ChAT was achieved by process-ing tissue for m2 staining with nickel II sulfate (blackreaction product) followed by ChAT by using DAB (brownreaction product) in five aged control cases. To visualizethe relationships between GAL-immunoreactive fibers andm2 neurons within the nucleus basalis, sections wereprocessed for GAL immunoreactivity with the nickel-intensification procedure followed by m2 staining withDAB as the chromagen. These procedures are modifica-tions of previously reported protocols that result in aneasily identifiable, two-color immunohistochemical profile(Mufson et al., 1989a, 1993).

Immunohistochemical controls

Immunocytochemical controls consisted of processingboth sections as described above, except that the primaryantibody solvent or an irrelevant IgG was substituted forthe primary antibody. In addition, control material wasobtained by absorbing 0.0012 mg/ml primary ChAT antise-rum with purified ChAT enzyme (Bruce et al., 1985;Mufson et al., 1988a,b), and the GAL antibody was preab-sorbed with 1 mM or 5 mM of the synthetic GAL peptide(Peninsula Laboratories) 24 hours prior to tissue incuba-tion. The potential for the antibodies to react with structur-ally related proteins, which is inherent to all immunohisto-chemical procedures, still cannot be excluded. Thus, adegree of caution is warranted, and m2, ChAT, p75NTR, andGAL immunostaining in this report, therefore, refers to‘‘-like’’ immunoreactivity.

Nissl cytochemistry

Additional sections were stained with thionin for cytoar-chitectonic delineation. In this and all other conditions,sections were dehydrated with graded alcohols, cleared inxylene, coverslipped with Permount (Fisher Scientific Co.),and analyzed microscopically under brightfield illumina-tion.

Morphometric analysis of immunopositiveprofiles

Sections throughout the human nucleus basalis wereevaluated for relative numbers of m2-, p75NTR-, ChAT-, anddouble-labeled (m2/ChAT) positive neurons by using acomputer-assisted morphometric system modified from apreviously reported procedure (Mufson et al., 1989a). Thedistribution and counts of m2-, ChAT-, and p75NTR-containing neurons were determined with the aid of aHewlett-Packard X-Y plotter (Corvallis, OR) attached to aDiLOG 40001 multimarker computer system (DiLog Inst.,Tallahassee, FL) coupled to the mechanical stage of aNikon microscope (Tokyo, Japan). The profile counts wereperformed at 2003 (103 oculars and 203 objective) magni-fication. Seven sections per case, containing the full extentof the nucleus basalis (Ch4), were evaluated from a 1-in-18series. Adjacent series of sections were processed for m2and the other markers. Absolute quantitation with unbi-ased methods (Coggeshall and Lekan, 1996) was not our

Fig. 2. Color photomicrographs of sections individually stainedand dual stained for m2 and choline acetyltransferase (ChAT) and m2and galanin (see text for double-labeling procedures). A: Photomicro-graph showing only two single-labeled m2 neurons (black) scatteredwithin a cluster of ChAT-positive perikarya (brown; arrows).B: Dual-stained sections of the striatum showing that virtually allChAT (brown) neurons also contain m2 immunoreactivity (black).C: Examples of single-stained m2 nucleus basalis neurons treatedwith nickel, resulting in a black reaction product. D: Section dualstained for ChAT (brown; open arrows) and m2 (black) within thenucleus basalis. Note the two large double-stained neurons and aChAT single-labeled neuron (solid arrows). E: Section dual stained forChAT (brown) and m2 (black) within the diagonal band region. Notethe double stained neuron (black and brown) and a ChAT single-labeled neuron (arrow). F: Section dual stained for ChAT (brown) andm2 (black) showing an m2-stained fiber coursing through a cluster ofcholinergic neurons. Note the close apposition of the m2 fiber (arrows)to the cholinergic perikarya. G,I: Sections dual stained for m2 (brown)and galanin (black). Note the decoration of galaninergic fibers on anm2 bipolar (G) and multipolar (I) neuron. H: Single-labeled galanin-immunoreactive neuron within the nucleus basalis. Scale bars 5 100µm in A, 50 µm in B–I.


objective because of recognized methodological limitations(see, e.g., Rouse and Levey, 1996). The high density ofneuropil (i.e., dendrites, axons, and terminals) m2 immu-nostaining may have resulted in some cases in an underes-timation of immunoreactive cell soma profiles. Further-more, the specimens used in this study were not preparedfor use with an optical dissector system. Thus, the dataprovided an indication of the relative differences in m2-labeled neurons between aged controls and AD brains.Statistical evaluations were performed by using a pairedt-test. The Ch1–Ch4 nomenclature (Mesulam et al., 1983;Mufson et al., 1989a) was applied to the human basalforebrain subfields.

RESULTS

m2- and ChAT-immunoreactive profilesin the aged human basal forebrain

In all aged human cases, dense accumulations of m2immunoreactivity were seen within the basal forebrain,including the medial septum, the vertical and horizontallimb nuclei of the diagonal band, the nucleus basalis-substantia innominata complex, and the bed nucleus of thestria terminalis (Figs. 1–4). The m21 neuronal stainingusing nickel intensification of the DAB chromagen yields ablue-black reaction product, which was present on the cellsurface and within the cytoplasm and proximal dendrites.Immunohistochemical control sections processed withoutthe primary antibody solvent or with the substitution ofirrelevant IgG failed to reveal any perikaryal or fiberstaining.

The cellular and regional distributions of m2-containingprofiles were compared with those expressing p75NTR (anexcellent marker for cholinergic neurons; see, e.g., Mufsonet al., 1989a) and ChAT immunostaining in adjacent (orclosely matched) sections in order to determine the relation-ship of the receptor protein to cholinergic neurons withinthe basal forebrain. In each case, dense accumulations ofm2 immunoreactivity were seen within processes andpuncta of the basal forebrain subfields (Figs. 1, 3B,D,4B,D). Many m2-immunostained fibers were seen in closeapposition to vascular elements (Fig. 5D). Embeddedwithin the densely stained m2 neuropil of the basalforebrain, there were scattered small, medium, and largemultipolar neurons (average size, 355.7 6 15.4 µm2; basedon an analysis of at least 100 m2 neurons) with longdendrites (Figs. 3D, 4D). The m2 immunostaining withinthe basal forebrain neuropil decreased in intensity in themore caudal regions of the nucleus basalis complex. Thisreduction in m2 immunoreactivity was most striking inthe intermediate and posterior aspects of the nucleusbasalis, making it easier to visualize receptor-positiveneurons. m2 receptor-containing neurons were also seenwithin adjacent hypothalamic regions (Figs. 6, 7). Incontrast, p75NTR and ChAT immunostaining revealed acontinuum of large multipolar neurons varying in shapefrom oval to round to triangular (average size, 538 6 12.8µm2; S.E.M. based on an analysis of at least 100 neuronscontaining each marker) that formed more well-definedcell aggregates within the various subfields of the nucleusbasalis (Figs. 2C, 3A,C, 4A,C). These cholinergic cell markersyielded a reaction product that is present on the cell

surface and within the cytoplasm and proximal dendrites(Figs. 2A,C, 3C, 5B). In contrast, sections reacted forp75NTR did not stain striatal neurons or the neuropil of thebed nucleus of the stria terminalis (Fig. 1). Despite thesesimilarities, there were significant differences betweeneach marker. For example, most of the m2-immunoreac-tive neurons were located outside the large cholinergic cellaggregates seen within the nucleus basalis subfields (2Aand 6). However, a few were admixed within the choliner-gic cell groups (Figs. 2A, 6). Cell counts from adjacentstained sections revealed that the relative numbers ofp75NTR containing neurons were about ten times moreabundant than m2-immunostained neurons throughoutthe basal forebrain complex. It is important to bear inmind that the dense m2 neuropil staining (see Figs. 3B,4B) may have obscured the visualization of some positiveneurons within the subfields of the basal forebrain. Thus,on the basis of cell size, frequency, and distribution,m2-immunoreactive neurons exhibited differences fromcholinergic neurons identified by ChAT and p75NTR stain-ing.

Immunohistochemical colocalization of m2and ChAT

Concurrent visualization of m2 and ChAT in the samesections revealed limited colocalization throughout theCBF (Figs. 2A,C–F, 6) and also confirmed the differencesobserved in adjacent series stained singly for each markeralone. In dual-stained sections, neurons immunopositivefor ChAT revealed a brown reaction product homoge-neously throughout the cell body and processes, whereasm2 immunoreactivity appeared as a blue-black reactionproduct scattered within the somata and its processes.This dual-color immunohistochemistry allows for the easydetection of double-stained neurons. Neurons expressingeither marker alone were found in the same sections asdouble-stained perikarya (Figs. 2A, 6). Computer-assistedchartings of sections dual stained for m2 and ChATrevealed that the distribution of these proteins was similarthroughout the basal forebrain as well as the striatum(Figs. 6, 7). Cell counts from a representative aged control(Table 1, case 5) were performed to provide an estimate ofthe degree of colocalization. In the septal/diagonal bandcomplex (Ch1-Ch2), m2 was detected in only 14% ofChAT-labeled neurons, whereas only 13% of m2-stainedneurons were dual labeled for ChAT. Within the nucleusbasalis, ChAT was found in 35% of the m2-labeled neu-rons, whereas only 6% of ChAT-stained perikarya weredual labeled for m2. These should be taken as minimumestimates, because visualization of the antigen may conceiv-ably limit detection of the other. In many instances, wealso observed m2-containing fibers coursing for long dis-tances within the cholinergic cell groups of the basalforebrain (Fig. 2F).

The striata from the same sections were analyzed for thelocalization of m2- and/or ChAT-immunoreactive immuno-staining for comparison, because the components of thestriatum contain intrinsic cholinergic perikarya and m2-containing neurons (Hersch et al., 1994; Levey et al.,1995). Within the striatum, we observed m2-immunoreac-tive, medium- to large-sized multipolar neurons scatteredthroughout the caudate and putamen (Figs. 2B, 5A, 6, 7).


Fig. 3. A,B: High-power photomicrographs of the diagonal bandregion shown in Figure 1A,D immunostained for p75NTR and m2,respectively. In A, note that the cholinergic marker distinguishes themedial and lateral divisions of the diagonal band more clearly.C: Photomicrograph of numerous p75NTR-immunostained cholinergic

diagonal band neurons and processes. D: In contrast, very few m2neurons were seen embedded within the immunoreactive neuropil ofthe diagonal band. Note the extensive dendritic process emanatingfrom the m2 neuron. Scale bars 5 500 µm in A,B, 100 µm in C,D.

The neuropil of the striatum was densely immunoreactivefor ChAT and m2 (Figs. 1, 2B). In addition, the nucleusaccumbens and the olfactory tubercle also contained m2immunopositive neurons. Dual-stained sections for thevisualization of m2 and ChAT revealed that the twomarkers were colocalized in the majority of striatal neu-rons (Figs. 2B, 6). Cell counts demonstrated that 90% ofChAT-labeled striatal neurons contained the m2 receptor.On the other hand, there were a few m2 neurons that didnot contain ChAT.

Colocalization of m2 and GAL

We identified a distinct subset of m2-immunoreactiveneurons in the basal forebrain that does not containdetectable levels of the cholinergic enzyme ChAT (Fig.2G–I).Although many of these perikarya displayed morpho-logical characteristics similar to the majority of cholinergic/m2-containing neurons, their precise chemical phenotypeis unknown. Because we (Kordower and Mufson, 1990;Benzing et al., 1993; Mufson et al., 1993) and others(Chan-Palay, 1988) have described in humans a subgroupof small to medium-sized neurons within the basal fore-brain that contain the neuropeptide GAL, which inhibitscholinergic neurotransmission (Fisone et al., 1987), weused dual-color immunohistochemistry to concurrentlystain sections for m2 and GAL (Fig. 2G–I). Evaluation of

this tissue revealed distinct populations of neurons contain-ing either GAL or the m2 receptor (Fig. 2G–I). We did notobserve any m2 neurons that coexpressed GAL, or viseversa. In contrast, GAL-containing fibers were seen tosurround m2-containing neurons (Fig. 2G,I) in a mannersimilar to that reported to occur in relation to cholinergicneurons in the human basal forebrain (Chan-Palay, 1988;Mufson et al., 1993).

m2 Immunoreactivity in AD

The cholinergic neurons of the basal forebrain undergoextensive cellular degeneration, resulting in a massive cellloss in AD (Whitehouse et al., 1981; Mufson et al., 1989b).Sections throughout the CBF from patients with AD wereimmunoreacted for m2, and the morphology, distribution,and numbers were compared with aged controls. Althoughmany m2 neurons exhibited morphological profiles similarto those seen in the aged brain (Fig. 8C), numerousreceptor-containing perikarya appeared shrunken withblunted dendrites in AD (Fig. 8D–G). Furthermore, therewas a tendency for a reduction in m2 neuropil stainingwithin the anterior regions of the basal forebrain in AD(Fig. 8A). m2 fibers often appeared thicken in the AD basalforebrain (Fig. 8B). Chartings of m2-containing neuronswithin the basal forebrain from normal aged and AD casesrevealed no disease-related difference in the distribution of

Fig. 4. A,B: High-power photomicrographs of the anterior medialregion of the nucleus basalis shown in Figure 2B,E immunostained forp75NTR and m2, respectively. The arrows in A and B indicate theoverlap between cholinergic and m2 staining in this region. Thearrowheads in A indicate the caudal extension of the diagonal bandnucleus. C: Photomicrograph of numerous p75NTR-immunostained

cholinergic nucleus basalis neurons. Note their magnocellular appear-ance and extensive dendritic processes. D: In contrast, very few m2neurons were seen embedded within the immunoreactive neuropil.Arrows in D indicate the location of m2-immunostained fibers. Scalebars 5 500 µm in A,B, 100 µm in C,D.


receptor-expressing perikarya (Fig. 7). Cell counts of therelative number of m2-immunoreactive neurons withinthe nucleus basalis between aged controls and AD failed toreveal significant differences between groups (Fig. 9; t 50.504; P 0.640).

DISCUSSION

The present investigation has revealed several majorfindings related to the cellular distribution of the m2mAChR protein in the basal forebrain of aged normalhumans and individuals with AD. Employing single- anddouble-immunocytochemical procedures, we have foundthat the m2 receptor protein is expressed both in asubpopulation of cholinergic perikarya and in many smallerneurons that were noncholinergic. In fact, the majority ofCBF neurons do not contain detectable levels of the m2receptor. For example, within the septal/diagonal bandcomplex and the nucleus basalis, approximately 13% and35% of ChAT-containing neurons coexpress m2, respec-

tively. In contrast to the extensive distribution of ChAT-containing neurons within the basal forebrain, the m2receptor protein is localized predominantly within theneuropil surrounding these cholinergic perikarya. It hasrecently been shown that much of the m2 receptor proteinfound within the neuropil of the rat septal/diagonalband complex is present in dendrites and in axon termi-nals (Levey et al., 1995). A comparison of the cellulardistribution of m2 within the aged normal and AD basalforebrain did not reveal differences in their location ornumbers. However, many m2 neurons appeared shrunkenwith blunted dendrites in AD. Furthermore, there was aminor reduction in m2 neuropil staining within the ante-rior regions of the basal forebrain in AD. Taken together,these observations suggest that the m2 muscarinic recep-tor plays a role in both cholinergic and noncholinergicsystems and that the cellular expression of thisreceptor is not significantly altered within the basal fore-brain in AD.

Fig. 5. Photomicrographs of m2 neurons and fibers. A: Immunore-active m2 multipolar neurons within the putamen. B: Large multipo-lar m2 neuron embedded within the fibers of the internal capsule.C: An m2-immunoreactive neurons within the nucleus basalis that

displayed a rounded cell body and a thickened dendritic branchingpattern. D: Numerous m2-immunopositive fibers in close apposition toa vascular element. Scale bars 5 50 µm in A–C, 100 µm in D.


Figure 6

m2 Receptor and CBF neurons

The present immunohistochemical findings demonstrat-ing the presence of m2 receptor protein in only a subset ofCBF neurons in humans are similar to reports in therodent (Levey et al., 1995) and monkey (Mufson et al.,1996a; Mrzljak et al., 1997). Recently, we have shown thatthe m2 receptor is present in the cell surface membranesat somata and dendritic postsynaptic sites as well as inaxons and axon terminals presynaptically (Levey et al.,1995). Our finding on the distribution of the m2 receptor inthe human basal forebrain is similar to the localization ofM2 radioligand binding sites described in this region inother mammals (Mash and Potter, 1985). Moreover, immu-noprecipitation studies also indicate that m2 is the mostabundant muscarinic receptor subtype in the human basalforebrain (Flynn et al., 1995). These findings suggest thatm2 protein is the molecular subtype corresponding to M2binding sites in the basal forebrain.

The fact that m2 receptor mRNA, protein, and M2binding sites are found within the basal forebrain lendssupport to the suggestion that this muscarinic subtype isan autoreceptor expressed in cholinergic neurons (forreview, see Levey, 1996). Support for this hypothesis in thehuman brain is gathered from our colocalization of m2 andChAT within in a subset of basal forebrain cholinergicneurons and also within virtually all striatal cholinergicperikarya. Cell counts in sections processed for the dualvisualization of these two antigens revealed that m2 isexpressed in at least 13% of cholinergic neurons within theseptal/diagonal band complex (Ch1-Ch2) and in 35% ofChAT-containing neurons within the nucleus basalis (Ch4).These numbers are very similar to those previously re-ported, demonstrating the relationship between m2 andcholinergic neurons within these regions in the rat (Leveyet al., 1995). One should bear in mind that it is likely thatsome cholinergic neurons are falsely negative for m2 in thepresent study due to cells containing levels of the proteinbelow the detectable limits of the current immunohisto-chemical procedure, obscured by the dense neuropil stain-ing, or because the majority of the receptor protein istransported away from the perikarya to presynaptic sitesin either the hippocampus or neocortex. Therefore, thenumbers presented in this study may be only minimalestimates, especially because concurrent labeling tech-niques are less sensitive than single-labeling methods(Levey et al., 1986). However, a comparison of cell countsderived from adjacent sections optimally prepared forsingle immunostaining for the basal forebrain cholinergiccell marker, p75NTR (Mufson et al., 1989a), and m2 re-vealed that cholinergic perikarya far exceeded the num-bers of m2 neurons within the human basal forebrain

(Mufson et al., 1996a; present study) but not in thestriatum. These observations support our findings in ratbasal forebrain (Levey et al., 1995). Immunoelectron micro-scopic studies indicate that some but not all m2 neuronsdisplay ultrastructural features consistent with perikaryacontaining ChAT in the rat basal forebrain (Dinopuolos etal., 1986; Levey et al., 1995). Taken together, these find-ings indicate that only a subgroup of cholinergic neuronsexpresses detectable levels of the m2 autoreceptor. Interest-ingly, these findings suggest that ACh release from variouscholinergic neurons may be differentially regulated. Alter-natively, only some cholinergic neurons may be responsivepostsynaptically to locally released ACh.

The present findings demonstrating that only a subset ofCBF neurons express m2 are surprising in light of earliersuggestions that these cholinergic cell bodies provided themajor source of the presynaptic m2 receptor within thecortex, hippocampus, and other terminal fields (Mash etal., 1985). However, the minor number of m2/cholinergicneurons found in the human (present study) and rat(Levey et al., 1995) basal forebrain argues against thishypothesis. Moreover, nearly complete lesions of ChAT-immunoreactive neurons with 192 IgG-saporin immuno-toxin resulted in little, if any, apparent reduction in m2expression within rat basal forebrain (Levey et al., 1995)and monkey cortex (Mrzljak et al., 1997). The preservationof m2 receptor activity following immunolesioned basalforebrain further suggests that numerous m2 neurons anddendrites in the basal forebrain are noncholinergic struc-tures. These observations may help explain the ratherminor reductions in presynaptic receptors determined byM2 binding or immunoprecipitation following lesion of thebasal forebrain projections (Wall et al., 1994; Levey et al.,1995).

m2 Noncholinergic neurons within the basalforebrain

Our investigation shows that m2 receptors are alsoexpressed in noncholinergic neurons within the basalforebrain. The m2-immunoreactive neurons have a similardistribution, but they are generally smaller and lessabundant than the cholinergic neurons. The chemicalphenotype of these noncholinergic neurons remains to bedefined. Previously, it has been suggested that some ofthese may be g-aminobutyric acid (GABA)ergic neuronsthat project to the hippocampus (Kohler et al., 1984;Fruend and Antal, 1988). It remains to be determine whichother peptides these noncholinergic m2 neurons contain.Several other possible candidates include members of thecalcium-binding protein family (Geula et al., 1993), somato-statin-like proteins (Mufson et al., 1988a), and NADPH-diaphorase-containing neurons (Geula et al., 1993; Benz-ing and Mufson, 1995), all of which are expressed byperikarya of the basal forebrain. In the present study,sections dual immunostained for m2 and the inhibitoryneuropeptide GAL did not reveal colocalization of theseproteins. Instead, GALergic fibers were seen in closeapposition to neurons and processes immunoreactive form2. The cells of origin of these GALergic fibers areunknown, but they are most likely derived from twosources: 1) local circuit neurons intrinsic to the basalforebrain (Chan-Palay, 1988; Mufson et al., 1993) or 2)extrinsic sources, including the locus coereulus (Chan-Palay, 1988) or the amygdala (Mufson et al., 1993). Be-cause we did not perform triple-label immunohistochemi-

Fig. 6. A–F: Schematic diagrams illustrating the distribution ofm2 (red dots), choline acetyltransferase (ChAT; purple dots), andChAT/m2 double-labeled (blue dots) neurons within the aged humanforebrain. Each dot represents five neurons. AC, anterior commissure;CD, caudate; Ch2, cholinergic cell group of the vertical limb of thediagonal band; Ch4am, cholinergic cell group, anteromedial; Ch4al,cholinergic cell group, anterolateral; Ch4i, cholinergic cell group,intermediate; Ch4id, cholinergic cell group, intermediate dorsal; Ch4iv,cholinergic cell group, intermediate ventral; Ch4p, cholinergic cellgroup, posterior; GP, globus pallidus; GPe, globus pallidus external;GPi, globus pallidus internal; hy, hypothalamus; IC, internal capsule;INS, insula cortex; LV, lateral ventricle; NAc, nucleus accumbens; OT,optic tract; PT, putamen.


Fig. 7. A–L: Schematic drawings comparing the cellular distribu-tion of m2-immunoreactive neurons within the forebrain of a normalaged control (AGED CONT.) and Alzheimer’s disease (AD) brain. AC,anterior commissure; BNST, bed nucleus of the stria terminals; CD,caudate; Ch2, cholinergic cell group of the vertical limb of the diagonalband; Ch4am, cholinergic cell group anteromedial; Ch4i, cholinergic

cell group, intermediate; Ch4p, cholinergic cell group, posterior;Ch4al, cholinergic cell group, anterolateral; fx, fornix; GP, globuspallidus; GPe, globus pallidus external; GPi, globus pallidus internal;hy, hypothalamus; IC, internal capsule; INS, insula cortex; LV, lateralventricle; Mb, mammilary body; NAc, nucleus accumbens; ot, optictract; PT, putamen; Th, thalamus.


Figure 7. (Continued)


Fig. 8. Photomicrographs of m2-immunostained profiles in ADbrain. A: M2 neuropil staining in the anteromedial-anterolateralsubdivisions of the nucleus basalis. Compared with the stainingpattern seen in aged controls (see Fig. 1F), there is a slight reductionin neuropil immunoreactivity for m2 in AD material. B: Example of athickened m2-immunoreactive fiber in AD brain. C,E: Healthy appear-

ing m2 immunoreactive neurons within the nucleus basalis andseptal/diagonal band region, respectively. D,F: Examples of shrunkenm2 neurons with blunted dendritic arbors. G: Higher magnificationphotomicrograph of the neurons shown in the lower left corner in F.Scale bars 5 500 µm in A, 50 µm in B,C,F, 100 µm in D,E, 25 µm in G.


cal experiments for ChAT, m2, or GAL, it remains to bedetermined whether the GALergic fibers are coursing inclose apposition to the m2/cholinergic neurons within thehuman basal forebrain. If GAL-containing fibers are syn-apsing upon m2 or m2/cholinergic neurons, then thissuggests that GALergic systems may also influence thepresynaptic regulation of m2 receptors.

m2 mAChR subtype in AD

The present study provides the first direct assessment ofthe cellular localization of m2-immunostained profiles inthe AD basal forebrain. Despite the fact that many m2neurons appeared shrunken with bunted processes, therewas a nonsignificant reduction in number of receptor-containing neurons in AD. This is in marked contrast tothe well-documented reduction in the larger cholinergiccortical projection neurons within the basal forebrain inAD (Whitehouse et al., 1981; Mufson et al., 1989b), whichwas also seen in the present cases. The differential vulner-ability between the magnocellular cholinergic and m2neurons within the basal forebrain is not unique, becausesmall neurons containing somatostatin (Mufson et al.,1988a) and NADPH-diaphorase are also preserved in AD(Benzing and Mufson, 1995) compared with the largercholinergic neurons. The mechanism(s) underlying thisapparent selective vulnerability remains to be determined.In this vein, it is interesting to note that CBF projectionneurons contain neurofibrillary material (Mufson et al.,1989b), whereas m2 as well as other smaller perikarya arevirtually tangle free in AD (unpublished observations).

Alternatively, all types of cholinergic neurons may bevulnerable, but those expressing m2 may be such a minor-ity of the total population of m2/cholinergic and noncholin-ergic cells that the reduction may not reach significance.Another intriguing possibility is that the present findingsare consistent with the suggestion that, in AD, manycholinergic cells only shrink (Pearson et al., 1983; Rinne etal., 1987; Allen et al., 1988) and down-regulate ChATmRNA (Strada et al., 1992) and protein (Perry et al., 1982),possibly in response to loss of targets, neurotrophic factors(Mufson et al., 1995, 1996a,b), and other insults, such asamyloid deposition (Arendt et al., 1985), while maintain-ing their expression of m2. This suggestion is supported byfindings demonstrating that other presynaptic cholinergicmarkers are also not universally reduced in AD, includingthe high-affinity choline uptake system (Pascual et al.,1991) and the vesicular ACh transporter (see, e.g., Kish etal., 1990; Ruberg et al., 1990), all key molecules involved inthe regulation of ACh synthesis, storage, and release. Afew investigations have demonstrated a reduction of aputatively presynaptic muscarinic receptor subtype (M2)in AD (Mash et al., 1985; Quirion et al., 1986) consistentwith a loss of cholinergic terminals in this disease. How-ever, this observation is not consistently observed in AD(Caulfield et al., 1982; Quirion et al., 1986), and it is nowevident from molecular anatomical studies that this recep-tor is expressed in both cholinergic and noncholinergicneurons (Levey et al., 1995; present study). In fact, lesionsof the CBF neurons that spare the noncholinergic neuronshave little apparent effect on m2 receptor expressionwithin the cortex (Levey et al., 1995; Mrzljak et al., 1997).This suggests that most m2 receptors in this region arelocated on noncholinergic structures and that they are nota major source of m2 receptors found in the cortex. Thus,the reduced levels in the relative numbers m2 receptorseen in AD probably reflect changes in other neuronpopulations (Mrzljak et al., 1995; Mrzljak et al., 1993;Mufson et al., 1997) rather than a loss of m2 neuronswithin the basal forebrain in this disease.

Finally, the present findings have significant implica-tions for pharmacologic therapy. For example, an m2antagonist may be beneficial therapeutically in AD byaugmenting release of ACh from the subpopulation ofcholinergic neurons that contain m2. However, our dataalso suggest a more complex role for the m2 receptor in anoncholinergic population of basal forebrain neurons. Thedistribution of receptor in the somatodendritic domain ofthese neurons suggests a postsynaptic function. In addi-tion, m2 neuropil staining in human basal forebrain islikely to include presynaptic receptors, as previously shownin rodents (Levey et al., 1995). Thus, it is difficult to predictthe net effect of m2-selective drugs. Indeed, studies inrodents suggest that m2-like agonists may induce actionswithin the basal forebrain that are behaviorally relevantto functional recovery following damage to cholinergicsystems (Givens and Olton, 1995). Further studies willshed new light on the involvement of the m2 mAChR in ADand will provide future directions for the development ofnovel pharmacological treatments for this disease.

ACKNOWLEDGMENTS

We thank Drs. L.B. Hersh and M. Bothwell for thegenerous gift of the ChAT and p75NTR antibodies, respec-tively.

Fig. 9. Histogram showing that m2-immunoreactive neurons arenot reduced significantly within the nucleus basalis in AD materialcompared with aged controls.


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m2 muscarinic acetylcholine receptor-immunoreactive neurons are not reduced within the nucleus...

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