THE JOURNAL OF COMPARATIVE NEUROLOGY 330:15-31 (1993)

Heterogeneity and Selectivity of the Degeneration of Cholinergic Neurons in

the Basal Forebrain of Patients With Alzheimer's Disease

STEPHANE LEHERICY, ETIENNE C. HIRSCH, PASCALE CEKVERA-PI~ROT, LOUIS B. HERSH, SERGE BAKCHINE, FRANCOIS PIETTE,

CHARLES DUYCKAERTS, JEAN-JACQUES HAUW, FRANCE JAVOY-AGID, AND YVES AGID

INSERM U.289, BAtiment Nouvelle Pharmacie EL . , E.C.H., P.C.-P., F.J.-A,, Y.A.), Service de Neurologie (S.B.), and Laboratoire de Neuropathologie Robert Escourolle (J.-J.H., C.D.)

Hbpital de la Salp&trit.re, 47, boulevard de I'HBpital, 75013 Paris cedex 13, France; Service de Medecine Interne et GBriatrie, HBpital Charles Foix, 7, avenue de la republique, 94206 Ivry sur Seine, France (F.P.); Department of Biochemistry University of Texas Health Science

Center, Dallas, Texas 75235 (L.B.H.).

ABSTRACT Cholinergic neurons were studied by immunohistochemistry, with an antiserum against

choline acetyltransferase (ChAT), in the basal forebrain (Chl to Ch4) of four patients with Alzheimer's disease (AD) and four control subjects. ChAT-positive cell bodies were mapped and counted in Chl (medial septal nucleus), Ch2 (vertical nucleus of the diagonal band), Ch3 (horizontal nucleus of the diagonal band) and Ch4 (nucleus basalis of Meynert). Compared to controls, the number of cholinergic neurons in AD patients was reduced by 50% on average. The interindividual variations in cholinergic cell loss were high, neuronal loss ranging from moderate (27%j to severe (63%). Despite the small number ofbrains studied, a significant corre- lation was found between the cholinergic cell loss and the degree of intellectual impairment. To determine the selectivity of cholinergic neuronal loss in the basal forebrain of AD patients, NPY-immunoreactive neurons were also investigated. The number of NPY-positive cell bodies was the same in cont.rols and AD patients. The results (1) confirm cholinergic neuron degeneration in the basal forebrain in AD and the relative sparing of these neurons in some patients, (2) indicate that degeneration of cholinergic neurons in the basal forebrain contrib- utes to intellectual decline, and (3) show that, in AD, such cholinergic cell loss is selective, since NPY-positive neurons are preserved in the basal forebrain.

Key words: choline acetyltransferase, neuropeptide Y, immunohistochemistry, septal area,

c 19% Wiley-Lisa, Inc.

nucleus basalis of Meynert

Cholinergic neurons of the basal forebrain have been extensively studied in nonhuman animals (Kimura et al., '81; Fibiger, '82; Armstrong et al., '83; Houser et al., '83; Mesulam et al., '83a; Butcher and Woolf, '84) and primates (Hedreen et al., '83; Mesulam et al., '83b; Satoh and Fibiger, '85a,b) by neuronal tracing and choline acetyltrans- ferase ( C U T ) immunohistochemistry. These neurons form a cholinergic column comprising the medial septum, the nucleus of the diagonal band of Broca, and the nucleus basalis of Meynert. The medial septum and the vertical limb of the nucleus of the diagonal band provide the cholinergic innervation of the hippocampal formation; the horizontal limb of the nucleus of the diagonal band projects to the

olfactory bulb, and the nucleus basalis projects to the amygdaloid nuclei and the cerebral cortex (Swanson and Cowan, '79; Fibiger, '82; Mesulam et al., '83b). Mesulam et al. ('83a,b, '88) proposed the Ch nomenclature (Chl to Ch4) to designate the various subdivisions of the basal forebrain cholinergic neurons. In humans, several studies, by acetyl- cholinesterase (AChE) histochemistry or ChAT immunohis- tochemistry, have documented the organization of the cholinergic neurons of the nucleus basalis (Pearson et al., '83; Nagai et al., '83a; Hedreen et al., '84; Perry et al., '84; Saper and Chemlinski, '84; German et al., '85; Mesulam

Accepted October 28, 1992.

B 1993 WILEY-LISS, INC.

16 S. LEHERICY ET AL

and Geula, '88; Mufson et al., '89). More recently, the organization of CUT-immunoreactive neurons within the septum-diagonal band complex in humans has also been reported (Mufson et al., '89).

Degeneration of basal forebrain cholinergic neurons is a recognized neuropathological feature of Alzheimer's dis- ease (AD) (Whitehouse et al., '81; Nagai et al., '83b; Arendt et al., '85; Etienne et al., '86). The loss of cholinergic neurons in the nucleus basalis was first suggested following the recording of decreased choline acetyltransferase (CUT) activity in the cerebral cortex and the hippocampus (Davies and Maloney, '76; Perry et al., '78; Reisine et al., '80; Rossor et al., '82; Bird et al., '83; Henke and Lang, '83; Etienne et al., '86). CUT-positive neuropil is also decreased in the hippocampus (Ransmayr et al., '89). Neuronal loss was subsequently reported in the basal forebrain, as studied with the cresyl violet technique (Whitehouse et al., '81; Tagliavini and Pilleri, '83; Arendt et al., '83, '85; Etienne et al., '86). Few immunohistochemical studies have confirmed cholinergic cell loss in the nucleus basalis by means of antibodies against ChAT (Nagai et al., '83b; Mesulam and Geula, '88). Studies have also reported cell loss of nerve growth factor receptor (NGFR)-immunoreactive neurons in the nucleus basalis and the septum-diagonal band com- plex (Kordower et al., '89). In these structures, 95% of NGFR-positive neurons contain the cholinergic marker ChAT (Kordower et al., '89; Mufson et al., '89). However, to our knowledge, no study of the cholinergic neurons in the septum-diagonal band complex by means of C U T immuno- chemistry has previously been reported in AD.

The loss of cholinergic neurons in the basal forebrain varies from one patient to another, ranging from 18% to severe (Whitehouse et al., '81; Arendt et at., '83, '85; Nagai et al., '83b; Pearson et al., '83; Tagliavini and Pilleri, '83; Etienne et al., '86). Apart from interindividual variations caused by differing susceptibility of patients to the degener- ative process, this broad spectrum of results may stem from differences in the method used: for example, the limited number of brain sections studied, and the variable degrees

of cholinergic neuronal shrinkage, which may interfere with the size criterion for neurons to be counted by the cresyl violet technique (Pearson et al., '83; Tagliavini and Pilleri, '83; Arendt et al., '83, '86; Etienne et al., '86; Vogels et al., '90). Finally, differences in cell loss may depend upon the identity of the cells counted. The facts that normal concentrations of neuropeptide Y (NPY) and somatostatin and elevated levels of galanin have been found in the nucleus basalis suggest that neurons containing NPY, somatostatin, and galanin are preserved in that structure (Beal et al., '90). However, the selectivity of cholinergic neuronal loss has yet to be proven.

In the present study, a qualitative and quantitative analysis of cholinergic neurons was performed by means of ChAT immunohistochemistry over the entire length of the septum-diagonal band-nucleus basali s (Ch 1-Ch4 complex) of controls and AD patients. The aim was: (1) to assess the vulnerability of the various subdivisions of cholinergic neurons in the basal forebrain of AD patients, (2) to investigate correlation between cholinergic neuron loss and varying degrees of intellectual impairment, (3) to investi- gate morphological changes of cholinergic neurons by mea- suring cross-sectional areas of immunoreactive cell bodies, and (4) to substantiate the selectivity of cholinergic neuro- nal loss in the basal forebrain of AD patients by investigat- ing NPY-immunoreactive neurons.

MATERIALS AND METHODS Subjects

The brains of four control subjects with no history of neurological or psychiatric illness and four patients with AD were studied (Table 1). All AD patients had been insti- tutionalized in a geriatric department (Charles Foix Hospi- tal, Ivry). Controls andAD patients were age-matched (con- trols = 87.8 & 1.5 years; AD patients = 89.5 ? 5 years). Time elapsed between death and fixation of the tissue was not significantly different between control and AD subjects

A ac AChE AD al aP BNST

CD ChAT Chl ChZ Ch3 Ch4 Ch4a Ch4am Ch4al Ch4ai

Ch4id Ch4iv Ch4p C1 CM fl ft fx GP

cc

Ch41

amygdaloid nuclei anterior commissure acetylcholinesterase Alzheimer's disease ansa lenticulai-is ansa pedoncularis bed nucleus of the stria terminalis corpus callosum caudate nucleus choline acetyltransferase medial septa1 nucleus vertical limb of the diagonal band horizontal limb of the diagonal band cholinergic components of the nucleus basalis anterior subsector of Ch4 anteromedial subsector of Ch4 anterolateral subsector of Ch4 anterointermediate subsector of Ch4 intermediate subsector of Ch4 intermediodorsal subsector of Ch4 intermedioventral suhsector of Ch4 posterior subsector of Ch4 claustrum corpus mamillare fasciculus lenticularis fasciculus thalamicus fornix globus pallidus

Abbreuratrons

GPe GPi GPv H HT ic LS lsa me mi Na Nm NPY Nva NVl o c ot P PG PO pta Ptl 5m SP st T V, vs

globus pallidus (external part) globus pallidus (internal part) ventral pallidum hippocampus hypothalamus internal capsule lateral septum lenticulostriate arteries external medullary lamina of the globus pallidus internal medullary lamina of the globus pallidus nucleus anterior thalami nucleus medialis thalami neuropeptide Y nucleus ventralis anterior thalamus nucleus ventrolateralis thaiami optic chiasm optic tract putamen parolfactif gyrus preoptic area pedonculus thalami anterior pedonculus thalami inferior stria medullaris septum pellucidum stria terminalis thalamus third vcntricule ventral striatum

DEGENERATION OF CHOLINERGIC NEURONS 17

TABLE 1. Characteristics of Patients

Clinical Duration Associated Postmortem Brain Senile plaques Mini mental pathology delay (hours\ weight (g) lmmL ' scnre Age

M Alzheimer .5 Meiena 9 1,150 > 50 0 101 F Alzheiiner 7 Bronchopneumonia 16 950 3U 0 93 Alrhrirner ? Renal failure 14 1,075 22 3 84 F Alzheimer 3 Bronchopneumonia 13 1,170 20 13

N (years) Sex diagnosis (years)

80 2 1

3 F 4 5 6 92 7 88 F Cnntrol - Bronchopneumonia 24 1,250 8

86 F Control - Breast carcinoma 48 1.150 4 29 7 - F Control ~ Pulmonary emhuiism 940 0

85 M Control ~ Bronchopneumonia 12 1.200 0 2 -

-

Lung carcinoma

'Temporal cortex.

(controls = 22.8 2 9.1 hours; AD patients = 13 2 1.5 hours, P = 0.33). AD was diagnosed clinically in accordance with NINCDS-ADRDA criteria (McKhann et al., '84). Neuro- psychological testing included mini mental status (MMS) (Folstein et al., '75) performed a maximum of six months before the death of the patient. Diagnosis was confirmed postmortem by counting silver-impregnated (Bodian) senile plaques (Lamy et al., '89) in the temporal cortex (Table 1) as previously described (Ransmayr et al., '89). Sections of the basal forebrain were also stained with hematoxylin-eosin to verify the absence of vascular lesions.

Tissue preparation The brains were obtained at routine autopsy and trans-

ferred from the autopsy room to laboratory at +4"C. The brains were hemisected and cut rostrocaudally in the coronal plane into 1.5 cm slabs. Blocks of tissue containing the caudate nucleus. the putamen, the septal region, the pallidum, the nucleus basalis, and the thalamus were fixed for three days in 4% parformaldehyde (wt/vol)/ 15% picric acid (vol/vol), as previously described (Lehericy et al., '89), frozen in powdered dry ice and cut into serial coronal sections (40 km thick) on a freezing microtome. The region studied extended from the anterior border of the septal nuclei to the mammillary bodies.

Immunohistochemistry ChAT immunohistochemistry was performed with a poly-

clonal antiserum against human C U T provided by Dr L.R. Hersh (Bruce et al., '85; German et al., '85). Sections taken at 720 pm intervals (20-25 sections per brain) were incu- bated for three days with the polyclonal antiserum. Immuno- labeling was revealed by the double bridge peroxidase antiperoxidase (PAP) method (Graybiel et al., '87). The dilution of the antiserum was 1200 (Lehericy et al., '89). Absorption of the antiserum with 48 pM human placental ChAT (Sigma C2898) completely abolished the immunore- activity (Lehericy et al., '89). One section from each brain was processed without primary antibody; no staining was observed under these conditions.

NPY immunohistochemistry was performed on adjacent sections with an antiserum against NPY, kindly provided by Dr P.C. Emson (Institute of Animal Physiology, Cam- bridge, U.K.) (Dawbarn et al., '84). The dilution of the antiserum was 1:250 (Lehericy et a]., '89). Sections were incubated for three days with the antiserum and revealed by the PAP method.

Histochemistry Sections taken at 360 pm intervals were also processed

by AChE histochemistry (Graybiel and Ragsdale, '78).

Sections were selected so that one AChE section out of two was adjacent to a section stained with ChAT antibody.

Morphological analysis To determine whether the size of the C U T - or NPY-

positive neurons was modified in AD patients, the cross- sectional surface areas of 50 (at the level of Chl, Ch2 and Ch31, 150 (at the level of Ch4) CUT-positive cells and 50 NPY-positive cells (at the level of the anterior commissure) were measured in two control subjects and two AD patients.

Quantification of immunostained cell bodies ChAT-stained cell bodies in each of the sections from

each patient were mapped by an operator with the aid of a semi-automatic image analysis system (HISTO-RAG, Bio- corn, France). Every CUT-positive cell body, displaying either strong or light immunostaining, was mapped and counted. The cholinergic cell complex was then subdivided according to the nomenclature of Mesulam et al. ('83b, '88). For each control subject and AD patient, the number of cells per section was plotted against the rostrocaudal loeation of the section. The area under the resulting curve was used to estimate the total number of ChAT-immuno- reactive cell bodies in each region studied. The mean %

standard error of the mean (S.E.M.), in arbitrary units, was then calculated for controls and AD patients. The total number of ChAT-positive cells was estimated: (1) for the entire length of the structure (Chl-4); (2) for Chl (medial septal nucleus), Ch2 (vertical limb of the diagonal band), both separately and together (Chl-Z), Ch3 (horizontal limb of the diagonal band) and Ch4 (nucleus basalis); (3) for the main subdivisions of Ch4: anterior (Ch4a), anterointerme- diate (Ch4ai), intermediate (Ch4i) and posterior (Ch4p); and (4) for each of the subdivisions of CMa, medial (Ch4am) and lateral (Ch4al), and the subdivisions of Ch4i, ventral (Ch4iv) and dorsal (Ch4id). Boundaries between the different subdivisions of the Chl-Ch4 complex were chosen as follows. The demarcation between Chl and Ch2 was determined by a rarefaction of neurons (Fig. 1B-D). Ch4a was situated immediately posterior to the olfactory tubercle (Fig. 2A). The Ch4am subsector was separated from Ch4al by a vascular structure or a rarefaction of neurons usually in the middle of the ventral portion of the pallidum (Fig. 2A-C). This separation was less clearly defined in AD brains since neurons were sparse. The division was there- fore considered to correspond to the middle of the ventral portion of the pallidum. Ch3 was considered to be the most ventral part of the nucleus basalis, located beneath the cholinergic neurons of Ch4 (Fig. 2C,D; 3A). The Ch4i subsector was delineated by the ansa peduncularis (ventral amygdalofugal pathway) dividing Ch4i into a ventral (Ch4iv) and a dorsal (Ch4id) compartment (Fig. 3B,C). More cau-

18 S. LEHERICY ET AL

Fig. 1. A-D: Maps of CUT-positive neurons in serial 40 pm coronal sections of the basal forebrain in a control patient. The angle of the coronal plane was chosen as close as possible tn the angle of coronal figures in Nieuwenhuys et al. ('88). The drawings are based on the

results obtained in control No. 8. Sections are disposed along the rostrocaudal axis of Chl and Ch2. Sections are separated by 1,440 pm. Each dot represents one CUT-positive neuron. Scale bar = 3 mm and applies to A-D.

dally, Ch4p was located posterior to the passage of the ansa peduncularis (Fig. 3D). No cell counting was performed caudal to the level of the mammillary bodies, first because tissue was not available for all control subjects, and second,

because ChAT-positive cell bodies were not present in sufficiently large number for quantitative comparison. The mean number of CUT-positive cell bodies for the entire Chl-Ch4 complex was also estimated in controls by interpo-

DEGENERATION OF CHOLINERGIC NEURONS 19

A - Fig. 2. A-D Maps of ChAT-positive neurons in serial 40 pm

coronal sections of the basal forebrain in control patient No. 8 ithe same as in Fig. 1). Sections are disposed along the rostrocaudal axis from the anterior pole of Ch4 to Ch4ai. Sections are separated by 1,440 pm

except at the level of the anterior commissure, where they are separated by 720 pm (A and B). Each dot represents one CUT-positive neuron. Scale bar = 3 mm and applies to A-D.

20 S. LEHERICY ET AL

Figure 3

DEGENERATION OF CHOLINERGIC NEURONS 21

Fig. 4. CUT-containing neurons in controls. A: ChAT-immunostained neuron in Chl . B: ChA’r- immunostained neuron in Ch2. C: ChAT-immunostained neuron in Ch3. D: ChAT-immunostained neuron in Ch4. Scale bar = 30 pm and applies to A-D

lating the number of neurons per section over the entire length of the structure. For each control subject, the value was corrected for split cell counting error (Weibel, ’79). Values for each region were compared with the Student’s t-test.

NPY-positive cell bodies were also mapped and counted along the rostrocaudal axis of the Chl-Ch4 complex. Boundaries between the subdivisions were transferred from adjacent CUT-stained sections. Statistical analysis of NPY-positive cell bodies was performed only at the levels (Ch4a, CMai, and Ch4i) where such cells were present in sufficiently large numbers. Values for each region were compared with the Student’s t-test.

RESULTS Distribution, morphology, and number of

choline acetyltransferase- and neuropeptide Y-immunoreactive cell bodies in controls

ChAT-immunoreactiue neurons. Cholinergic forebrain neurons formed a continuous column from the medial septum to the level of the lateral geniculate body, extending 18-22 mm rostrocaudally (Figs. 1-3). Caudal to the level of

Fig. 3. A-D Maps of CUT-positive neurons in serial 40 pm coronal sections of the basal forebrain in control patient (the same as in Figs. 1 and 2). Sections are disposed along the rostrocaudal axis from Ch4ai to Ch4p. Sections are separated by 1,440 pm. Each dot reprc- sents one CUT-positive neuron. Scale bar = 3 mm and applies to A-D.

the mammillary bodies, CUT-positive cell bodies in our brains were not present in sufficiently large number for quantitative comparison. These magnocellular neurons dis- played heterogeneous size and shape, and many contained lipofuscin (Fig. U-D), Immunostaining was variable, rang- ing from light to strong. Cholinergic neuropil was intensely stained in the Chl-Ch4 complex, although to a lesser extent than in the striatum (Figs. 5,6).

Cholinergic neurons in Chl were large (30-55 pm long axis, mean surface area 693 ? 24 Fm2, Table 2). They tended to be ovoid (Fig. 4A) with vertical orientation. Many were embedded among the fibers of the precommissural fornix.

Cholinergic cell bodies in Ch2 were large (30-60 pm long axis, mean surface area 698 ? 19 Fm2, Table 21, mostly multipolar (Fig. 4B), and lay within the ventral continuity of the medial septum (Figs. 1B-D; 5A). Caudally, Ch2 merged with the nucleus basalis at the point where it reached the posterior edge of the ventral striatum (olfactory tubercle, Fig. 2A).

These neu- rons tended to be more fusiform than Ch4 neurons (10-20 pm short axis x 40-50 pm long axis, mean surface area 668 ;t 50 km2, Table 2, Fig. 4C). Ch3 had even less clearly defined bondaries than the other subregions. These cells were located between the preoptic area medially and the amygdaloid area laterally at the ventral border of the substantia innominata (Figs. 2C,D; 3A). Cholinergic neu- rons in Ch3 were usually orientated parallel to the ventral

Chl-medial septa1 nucleus.

Ch2--vertical limb of the diagonal band.

Ch3-horizontal limb of the diagonal band.

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24 S. LEHERICY ET AL

surface of the brain. According to Mesulam et al. ('83b), only 1% of neurons in this nucleus are ChAT-positive.

Cholinergic neurons within the nucleus basalis-Ch4 complex were large (40-60 Lm long axis, mean cross-sectional area 856 ? 15 pm2, Table 2) heterogeneous in shape (round to oval, multipolar or more fusiform, Fig. 4D). These neurons were located immediately posterior to the olfactory tubercle (Ch4a) (Figs. 2A, 5B). In humans, the transition between Ch4a and Ch4i was found to be more gradual than in primates, giving rise to an anterointermediate subsector (Ch4ai) (Figs. 2D; 3 A 6A). Occasional cholinergic neurons morphologically similar to those of the nucleus basalis were encountered within adjacent fiber bundles (internal cap- sule, anterior commissure, ansa lenticularis, internal and external medullary laminae of the pallidum) (Figs. 2C,D; 3A-D). Cholinergic cell bodies were also encountered within the caudate nucleus, the putamen, the ventral striatum and the globus pallidus, mostly at the level of the anterior commissure for the latter (not shown).

In controls, the mean number of ChAT-positive neurons per section was 375 i 91 at the level of Chl and Ch2 (Fig. 7A), 530 * 133 at the level of the anterior commissure (Fig. 7B), 585 ? 96 at the level of Ch4ai (Fig. 7C),429 2 55 at the level of Ch4i (Fig. 7D), and 80 * 13 at the level of Ch4p (Fig. 7E). The mean number of ChAT-positive cell bodies was calculated to be about 100,000 cells over the length of the Chl-Ch4 cell complex. Variation in total cell counts (esti- mated by the area under the curve) between the four control brains was small.

iVPY-immunoreactiue neurons. NPY-positive cell bod- ies were smaller than ChAT-positive cell bodies (mean cross-sectional area: 267 ? 10 Km2, Table 2 ) . IVPY-positive neurons varied in shape, although the majority were ovoid or fusiform (Fig. 8A). In the rostra1 portion of the basal forebrain, NPY-positive neurons were located within the dorsolateral septum, usually lateral to Chl. Few were encountered within Ch2, the majority being located at the level of the anterior commissure, mostly in the lateral part of the nucleus basalis (Fig. 9A). Caudally, cell density diminished at the intermediate level (Fig. 9B). In the posterior part of the nucleus basalis, even fewer NPY- positive cell bodies were visible (Fig. 9C). At the level of the anterior commissure, NPY-positive neurons were also en- countered on the periphery of the pallidum and, more caudally, in the external and internal medullary laminae of the pallidum. The mean number of NPY-positive cell bodies was 68 ? 13 in controls at the level of the decussation of the anterior commissure (corresponding to CMa, Fig. 9A) and 31 2 9 at Ch4i level (Fig. 9B).

Ch4-nucleus basalis (Figs. 2; 3; 5B; 6).

Distribution, morphology, and number of choline acetyltransferase- and neuropeptide

Y-immunoreactive cell bodies in patients with Alzheimer's disease

ChAT-inmunoreactive neurons. In AD, CUT-positive neurons similar to those observable in controls with a large darkly stained cell soma and long neuritic processes were visible in the Chl-Ch4 cell complex (Fig. IOA-C). Others displayed degenerative signs. They appeared either swollen or shrunken (Fig. 10D); some of them had vacuolated cytoplasm. They contained lipofuscin. In patients with AD, mean cross-sectional areas of ChA'1'-positive cell bodies were not significantly different from control values (675 2 23 Fm2, P = 0.75 in Chl; 707 ? 18, P = 0.59 in Ch2; 700 ?

TABLE 2. Mean Neuronal Cross-Sectional Surface Area of ChAT- and NPY-Positive Cell Bodies ! ~ r n 2 )

NPY-positive CUT-positive cell bodies cell bodies

(at the level Subjects Chl Ch 2 Ch3 Ch4 of Chla)

Alzlieimer 6 7 5 ~ 2 3 707i1.5 700i37 843112 279i 9 Control 693k24 698t19 G f i X i F j O 856i15 267 t10

37, P = 0.61 in Ch3; and 843 2 12 pmz, P = 0.51 in Ch4, Table 2).

The total number of neurons was reduced by 50% on average (Table 3, Fig. 7F-J) with a large variation between patients, the overall neuronal loss ranging from 27% (brain 4) to 63% (brain 1). The individual pattern of cell loss was variable in the different subdivisions of the Chl-Ch4 complex. In the most severe cases (for example, brain 2), cell loss within the different subdivisions of the Chl-Ch4 complex was, in general, homogeneous. However, in certain subregions, neuronal loss was more severe than average (Ch4p, brain 1) or moderate (CMid, brain 3) . In the least severe case, brain 4, the global cell count was only slightly lower than control values (Table 3). However, while certain subregions were only slightly affected (Ch4a, Ch4ai, and Ch4i), others showed alarge reduction in cell numbers (Ch2 and Ch4p). Lastly, the total cell counts in the Chl-Ch4 complex of AD patients correlated with the MMS (r = 0.97, P = 0.03 for Chl-4; r = 0.92, P = 0.07 for Ch4; and r = 0.1, P = 0.94 for Chl-2).

NPY-immunoreactiue neurons. Mean cross-sectional ar- eas of NPY-positive cell bodies in AD patients were not significantly different from those of controls (279 ? 9 pm2 at the level of Ch4, P > 0.05, Table 2, Fig. 8B). The mean number of NPY-positive cell bodies per section was 55 2 6 in AD patients at the level of the decussation of the anterior commissure (corresponding to ChQa, Fig. 9D) and 23 i. 6 a t Ch4i level (Fig. 9E). In AD patients, the estimated total number of NPY-positive cell bodies in the subregions studied was not significantly different from control values (Fig. 9D-F). Standard deviation was moderate at the level of the anterior commissure, indicating that differences between cell counts in the four control brains were small. However, a moderate nonsignificant 32% decrease ( P = 0.3) existed at the Ch4i level (at this level, control values were more dispersed, with a higher standard deviation).

DISCUSSION The basal forebrain is a complex area (Alheid and Hei-

mer, '88) and agreement concerning the definition of its various subdivisions is not general (Butcher and Semba, '89). Chl appears to correspond to the septal component of the nucleus of the diagonal band of Andy and Stephan ('68) and the medial septal nucleus of Gaspar et al. ('85). Ch2 corresponds to the tubercular component of the nucleus of the diagonal band of Andy and Stephan ('68) and the vertical limb of the diagonal band of Gaspar et al. ('85). No clear consensus exists concerning the horizontal limb of the

Fig. 7. Maps of CMT-positive neurons in the basal forebrain of control subject No 8 (A-E) and AD patient No. 2 (F-J) on matched sections a t the levcl of Chl-Ch2 (A, F), Ch4a (B, G), CMai (C, H), C M i (D, I), and Ch4p (E, J). Scale bar = 3 mm and applies to A-J.

Figure 7

26 S . LEHERICY ET AL

Fig. 8. NPY-containing neurons. A Control NPY-immunostained neuron at the level of CMa. B: Alzheimer NPY-immunostained neuron at the level of Ch4a. Scale bar = 30 Fm and applies to A and B.

diagonal band (Butcher and Semba, '89). In the present study, we followed the definition of Mesulam et al. ('83b, '88). The cholinergic component of the nucleus basalis is designated as the NB-Ch4 complex, whereas the term nucleus basalis is used to designate all the components of the nucleus (i.e. both cholinergic and noncholinergic neu- rons). The different subdivisions of the NB-Ch4 complex are not well delineated in nonprimate mammals and appear to apply only to the NB-Ch4 complex of primates and humans (Butcher and Semba, '89). However, even though divisions appear to exist within the basal forebrain, thus delineating the different subsectors, they are frequently not clear-cut. Easily reproducible arbitrary boundaries were sometimes needed, especially in AD brains, where severe cell loss was encountered, with regard to the Ch3 subsector and the transition zones between Chl and Ch2, Ch2 and Ch4a. Such boundaries were chosen after careful compari- son of the different control brain subsectors. Thus, because boundaries between Chl and Ch2 are sometimes arbitrary and because both subdivisions project to the hippocampus (Mesulam et al., '83b), they were also taken together (Chl-2). The possibility that cell counts within the subsec- tors were affected by the choice of arbitrary subdivisions cannot be ruled out.

Cholinergic cell bodies similar in shape to the cholinergic neurons of the nucleus basalis of Meynert are usually observed within adjacent fiber bundles (anterior commis- sure, internal capsule, internal and external medullary laminae of the pallidum, ansa lenticularis, ansa pedoncu- laris) and the globus pallidus, where these cells are believed to be misplaced NB-Ch4 neurons (Mesulam et al., '83b; Mesulam and Geula, '88; Satoh and Fibiger, '85a). Thus, cholinergic neurons of the NB-Ch4 complex have been considered as constituting an "open" nucleus. However, cholinergic neurons of the globus pallidus have been charac- terized according to their specific striatal afferents (Zabor- szky et al., '84). In control brains, the number of cholinergic neurons in the Chl-Ch4 complex was estimated to be 100,000 per hemisphere. Previous studies using the cresyl violet technique reported larger numbers: 139,000 neurons (McGeer et al.. '84) and 200,000 neurons (Arendt et al., '85), in the same structure. The difference with the present results could be explained by differences in the methods used and by age-related neuronal loss in the nucleus basalis (McGeer et al., '84; De Lacalle et al., '911, since control

subjects in the study by Arendt et al. ('85), which reported the highest number, were 20 years younger than those in the present study.

NPY-positive neurons were encountered in the dorsolat- era1 septal nucleus, the vertical limb of the diagonal band, and the nucleus basalis. No such cells were detected in the medial septal nucleus. In the nucleus basalis, neurons containing NPY-like immunoreactivity were mostly concen- trated in the anterior (particularly in the lateral subdivi- sion), anterointermediate and intermediate compartments of the nucleus basalis. NPY-positive cell bodies were smaller and more fusiform than cholinergic cell bodies. This sug- gests that they do not colocalize with cholinergic neurons. Data concerning NPY-positive cell bodies are consistent with previous studies in the human septal area (Gaspar et al., '87) and in the primate nucleus basalis (Walker et al., '89). Although their functions are not known, NPY-positive neurons could act as local circuit neurons regulating cholin- ergic activity (Tamiya et al., '91).

To our knowledge, the present study provides the first quantitative estimate of cholinergic cell loss in the Chl- Ch2 complex and Ch3 by means of ChAT immunohistochem- istry. Cholinergic neurons represent only 10% of the neuro- nal population of Chl and about 1% of Ch3, whereas they constitute up to 70% and 95% of the neurons in Ch2 and Ch4, respectively (Mesulam et al., '83b). Thus, the use of a selective cholinergic marker to quantify the degeneration of cholinergic neurons is more crucial in Chl and Ch3 than in the other areas of the Chl-Ch4 complex. Previous studies have reported loss of NGFR-immunoreactive neurons in Chl and Ch2, where 95% of NGFR-positive neurons con- tain the cholinergic marker ChAT (Kordower et al., '89). Since Chl and Ch2 provide the cholinergic innervation of the hippocampus (Mesulam et al., '83b), the decrease in cholinergic neurons in the Chl-Ch2 subsectors very likely corresponds to the decrease in density of cholinergic C U T - positive fibers in the hippocampus (Ransmayr et al., '89).

Estimates of the total number of CUT-positive neurons in the different subdivisions of the Chl-Ch4 complex provide new evidence of the high variability of cholinergic neuron degeneration in the basal forebrain in patients with AD. In the present study, all CUT-positive neurons were counted in each subdivision of the Chl-Ch4 complex (except the posterior part of Ch4p). Thus, the variability of the cholinergic neuronal loss has to be attributed, at least in

DEGENERATION OF CHOLINERGIC NEURONS 27

~~~ ~

with Alzheimer's disease No. 2 (D-F) on matched sections at the level of Ch4a (A, D), Ch4i (B, E), and Ch4p (C, F). Scale bar = 4 mm and applics to A-F.

part, to the disease process itself and not to methodological bias. Previous studies, using the cresyl violet technique, have reported considerable variability in the interindividual cholinergic neuronal loss in the basal forebrain (White- house et al., '81; Tagliavini and Pilleri, '83; Arendt et al., '83, '85; Etienne et al., '86). Additionally, the degree of cholinergic cell loss seemed to be correlated with the severity of the disease, the smallest decrease being found in the least affected patient (patient 4). Lastly, age at onset may also interfere with the severity of the neuronal loss, since difference in the intensity of loss of cholinergic neurons has been reported between early and late onset AD patients (Tagliavini and Pilleri, '83; Mann et al., '84). In the present study, no correlation existed between loss of cholin- ergic neurons and age. However, only a small sample was

studied and all patients were over 80 years of age. Results obtained with brain 4 confirm that degeneration of choliner- gic neurons of the basal forebrain in patients with definite AD can be moderate: cell number was reduced by only 25% in Ch4. Thus, patient 4 had cortical pathology characteris- tic of AD and no significant loss of cholinergic neurons in the NB-Ch4 complex. This result is consistent with the small, 18% decrease of CUT-positive neurons in the nucleus basalis reported by Pearson et al. ('83). However, only a few sections could be analyzed in that study. In the present study, while a small cell loss existed in Ch4 of brain 4, other subsectors, such as Chl-2 and Ch4p, were affected (53% loss in Chl-2, Table 3). Some subsectors seemed to be more severely lesioned in individual patients. Previous studies have also reported a large variability of cell loss

28 S. LEHERICY ET AL

Fig. 10. ChAT-containing neurons in AD patients. A ChAT- healthy appearance (A-C), while others were dystrophic (D). ChAT- positive neuron in D was shrunken in size with short neurilic exlen- sions. Scale bar = 30 Fm and applies to A-D.

iinmunostained neuron in Chl . B: CUT-immunostained neuron in Ch2. C,D: CUT-immunostained neuron in Ch4. Some neurons had a

TABLE 3. Percentage of Remaining ChAT-Positive Neurons in the Various Subdivisions ofthe ChlLCh4 Complex of Patients With Alzheimer's Disease Compared to Controls

N MMS C h l 4 Chl-2 Chl Ch2 Ch3 Ch4 Ch4a Ch4am Ch4al Ch4ai Ch4i Ch4iv Ch4id Ch4p

1 n 37 50 39 54 40 34 32 45 30 21 33 12 44 7 2 0 46 36 33 37 2R 48 49 49 47 44 59 43 57 44 3 3 46 46 58 40 49 48 52 BB 49 33 41 21 75 30 4 13 73 47 56 44 52 75 64 54 70 72 84 Bfi 98 35

m e a n ? S E M 5 0 + 8 4 5 ~ 5 4 7 ~ 6 4 4 2 4 4 9 2 4 5 1 ~ 9 4 9 ~ 7 5 4 ? 4 4 9 ? 8 4 3 1 1 0 5 4 1 1 1 3 4 ? 1 2 B R ? 1 1 2 9 r 8

P v * nb *I f * ** ** f * *.( ** xx ** **

"P < 0.05; ** = P < 0.01; ns = nonsignificant.

between subsectors (Arendt et al., '83, '85; Etienne et al., '86; Vogels et al., '90; Iraizoz et al., '91). In these studies, alternatively Ch4p (Arendt et al., '851, Ch4i (Etienne et al., '861, Ch4p (Vogels et al., 'go), and Ch4a (Iraizoz et al., '91) were the most severely affected subsectors. The difficulty of finding clear demarcations between several subregions may also interfere with the results. A larger number of cases in the early stages of the disease should be evaluated to determine whether some subsectors (for example Ch4p, which appears to be the most consistently severely affected) are involved first in the basal forebrain in AD. These data also show that investigation of the loss of cholinergic neurons in the Chl-Ch4 complex requires a study of the overall rostrocaudal extent of the structure and not only a small part. Lastly, the possibility that cholinergic neurons

are still present but no longer exhibit detectable levels of C U T cannot be ruled out.

A comparison of cholinergic cell loss in the entire Chl- Ch4 complex and the degree of intellectual impairment (MMS) revealed a clear correlation. The absence of signifi- cant correlation with the MMS for the Ch4 subregion may be explained by the small number of brains studied. Previ- ous studies have reported that the decrease in cortical C U T activity is correlated with the intellectual impair- ment as assessed by psychometric tests (Perry et al., '781, and with the degree of loss of large neurons in the nucleus basalis (Etienne et al., '86). Indeed, cholinergic neurons of the basal forebrain have been implicated in learning and memory processes (for review, see Richardson and DeLong, '88). These results provide further evidence that a subcorti-

DEGENERATION OF CHOLINERGIC NEURONS 29

AD patients. For example, the lowest level of ChAT activity was found in the frontal and the temporoparietal associa- tion areas, which are severely affected cortical areas in AD; and (3) at least two patients with AD (Pearson et al., '83, and patient 4 in the present study) showed little reduction in cell number in the NB-Ch4 complex. Thus, AD with neurofibrillary tangles and senile plaques in the neocortex can occur without a significant cholinergic neuronal loss in the NB-Ch4 complex.

Not all cholinergic nuclei are affected in Alzheimer's disease. Loss of cholinergic neurons has been described in the septum-diagonal band complex and the NB-Ch4 com- plex (Whitehouse et al., '81; Tagliavini and Pilleri, '83; Nagai et al., '83b; Arendt et al., '83, '85; Etienne et al., '86; Kordower et al., '89; the present study), the globus pallidus (Lehericy et al., '911, and in the ventral striatum (Lehericy et al., '891, where cholinergic neurons could be interneu- rons (Butcher and Woolf, '84). All these nuclei are con- nected to severely affected limbic or paralimbic structures of the brain (Swanson and Cowan, '79; Herzog and Kemper. '80; Mesulam et al., '83b; Hyman et al., '84; Alheid and Heimer, '88). Pearson et al. ('85) and Saper et al. ('87) have proposed that the mechanism of spreading of the disease process could be transsynaptic. This hypothesis is rein- forced by the fact that no cell loss has been reported in the caudate nucleus (Lehericy et al., '89), putamen (Nagai et al., '83b; Lehericy et al., '89), or brainstem cholinergic nuclei, such as the laterodorsal tegmental nucleus (Brandel et al., '91) and the tegmentopedunculopontine nucleus (TPP) (Zweig et al., '87; Mufson et al., '88), which are not connected to limbic areas. In that case, the fact that AD can occur without significant cholinergic neuronal loss in the NB-Ch4 complex is in agreement with the hypothesis that degeneration of cholinergic neurons in the basal forebrain is a secondary change (Pearson and Powell, '87). The possibility that the pathological process may be concomit- tent in cortical and subcortical structures (Mann et al., '86) cannot be ruled out, however. Lastly, the fact that the vulnerable cholinergic nuclei are connected to affected structures, such as the cerebral cortex, does not in itself seem to be a sufficient condition to lead to cholinergic cell death. For example, striatal GABAergic neurons, which are presumably spared in AD (Rossor et al., '82), receive direct connections from cortical glutamatergic neurons (see re- view in Smith and Bolam, '90). Other factors are probably implicated in the pathogenesis of cholinergic cell death. The selective vulnerability of specific subsets of cholinergic neurons has yet to be elucidated.

cocortical cholinergic deficiency contributes to the pathogen- esis of intellectual impairment in AD. However, in view of the severe histopathological changes found in the cerebral cortex and hippocampal formation, it is unlikely that cholinergic deficiency alone is responsible for the intellec- tual impairment.

The mean cross-sectional surface areas of cholinergic neurons were not significantly different in controls and AD patients in any of the subdivisions of the Chl-Ch4 complex. In Ch2 and Ch3, AD values were even slightly increased. A broad spectrum of morphological changes has been re- ported in the literature. Reduced (Pearson et al., '83), unchanged (Etienne et al., '86) or even increased (Arendt et al., '86) cross-sectional surface areas, decreased maximum diameter (Vogels et al., '901, increased cross-sectional nu- clear areas (Iraizoz et al., '911, and decreased nucleolar volume of cholinergic neurons (Tagliavini and Pilleri, '83; Mann et al., '84) has been reported in AD patients. In- creased cell and nuclear sizes have been interpreted as indices of regenerative change (Arendt et al., '86; Iraizoz et al., '91). In the present study, degenerative neurons were encountered, as well as healthy neurons. Some neurons were shrunken while others were enlarged. The data suggest that, although the mean cross-sectional surface area of cholinergic neurons may not be different from controls, both degenerative and regenerative changes are encountered in the basal forebrain ofAD patients. Compen- sation for cholinergic neuronal loss has been evidenced in AD. Increased levels of 3H-vesamicol, a marker of vesicular ACh reuptake in terminal fibers, have been reported in the hippocampus, suggesting that compensation for cell loss exists in this structure at the level of the vesicular reuptake system (Ruberg et al., '90). In line with this hypothesis, abnormal cholinergic nerve fibers with regularly spaced swellings (compatible with an increase in synaptic vesicles in the hypertrophied varicosities) have also been described in the hippocampus of AD patients (Ransmayr et al., '89).

The present study provides new evidence for the selective degeneration of cholinergic neurons in the basal forebrain of AD patients. Quantitative estimates of the total number of NPY-positive cell bodies at the level of Ch4a, Ch4ai, and Ch4i indicate the preservation of such neurons in the nucleus basalis. NPY-positive neurons in the lateral septa1 nucleus and the vertical limb of the diagonal band were too sparse for a quantitative comparison to be made. In the ventral striatum, where cholinergic neurons also degener- ate, NPY-positive neurons are likewise unaffected (Lehericy et al., '89), indicating that the latter are generally preserved in the basal forebrain of AD patients.

The mechanism of cholinergic cell death is still unknown. It has been hypothesized that loss of cortical cholinergic fibers is implicated in the pathogenesis of neuritic plaques in the cortex (Arendt et al., '85; Struble et al., '82). Evidence against a determining role for cholinergic fibers in the formation of senile plaques has been reported: (1) two studies failed to demonstrate any correlation between the distribution of senile plaques stained with thioff avine S and the loss of CUT-positive fibers in the different subregions of the hippocampus of AD patients (Ransmayr et al., '89, '91). This fact argues against a direct relationship between the degeneration of ChAT-positive fibers and the presence of senile plaques in the hippocampus of AD patients; (2) Mesulam et al. ('86) have shown that the regional distribu- tion of cholinergic innervation in primates does not corre- spond to the distribution of senile plaques in the cortex of

ACKNOWLEDGMENTS We thank Prs. J.P. Bouchon, R. Moulias, Drs. M. Laur-

ent, and A. Sachet, who contributed case material and Dr. P.C. Emson who provided NPY antiserum. The research was supported in part by grants from the Assistance Publique des HBpitaux de Paris (Contrat de recherche clinique No. 912208).

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