Choline acetyltransferase-like immunoreactivity in the hippocampal formation of control subjects and patients with Alzheimer's disease

0306-4522/89 %3.00 + 0.00 PergamonPressplc 0 1989IBRO

Neuroscience Vol. 32, No. 3, pp. 701-714,1989 Printedin Great Britain

CHOLINE ACETYLTRANSFERASE-LIKE IMMUNOREACTIVITY IN THE HIPPOCAMPAL FORMATION OF CONTROL SUBJECTS AND PATIENTS WITH ALZHEIMER’S DISEASE G. RANSMAYR,* P. CERVERA,* E. HIRSCH,* M. RUBERG,* L. B. HERsH,P C. DUYCKAERTS,~ J.-J. HAUW,$ C. DELUMEAU* and Y. ACID* *Laboratoire de Mtdecine Expkrimentale (INSERM U. 289), HBpital de la SalpitrZre, 47 Boulevard de I’Hbpital, 75013 Paris, France tDepartment of Biochemistry, University of Texas Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235-9083, U.S.A. SLaboratoire de Neuropathologie R. Escourolle, Hapita de la Salpi+tri&re,47 Boulevard de I’Hbpital, 75013 Paris, France Abstract-A qualitative and quantitative immunohistochemical study of cholinergic systems in the human hippocampal formation was performed with an antibody against choline acetyltransferase. Four control subjects and six patients with Alzheimer’s disease, matched for age and post-mortem delay, were examined. Immunoreactive nerve fibres and terminals were visualized, but no cholinergic cell bodies were seen. The distribution of the fibres and terminals suggests that a major afferent cholinergic pathway enters the hippocampus dorsally via the fimbria-fomix, a minor input entering from the temporal lobe along the alvear path. The cholinergic innervation suffers some degenerative change in normal aged subjects, but decreases considerably in density in patients with Alzheimer’s disease. The extent of the decrease differs somewhat among the subregions of the hippocampus, but is homogeneously distributed within each subregion, and throughout the rostrocaudal extent of the structure. Compensatory sprouting in reaction to denervation was not detected.

hippocampal formation, consisting of the hippocampus proper, the dentate gyrus and the subiculum,” with an antiserum specific for human ChAT to avoid the ambiguity of AChE staining, and to compare the normal pattern of innervation with that in hippocampus from patients with Alzheimer’s disease.

Cholinergic systems in the human hippocampus have been described until now by means of acetylcholinesterase (AChE) histochemistry.‘8,23*36,46,”The

enzyme is also found, however, in non-cholinergic neurons.24.28 To our knowledge, these systems have not been visualized in human brain with more specific cholinergic markers, although senile plaques have EXPERIMENTAL PROCEDURES been labelled in the hippocampus of patients with Alzheimer’s disease with an antiserum against choline Subjects acetyltransferase (ChAT).’ The brains of four control subjects and six patients with Alzheimer’s disease, exempt from vascular disease, brain Biochemical and anatomopathological studies tumors, brain-related infection, or psychiatric disorders not have shown that the activities of cholinergic marker related to Alzheimer’s disease, were studied (Table 1). All enzymes (ChAT and AChE) decrease in the neocorhad been institutionalized in a geriatric department (Charles tex as well as in the hippocampus of patients with Foix Hospital, Ivry), except patient No. 1 (Department of Alzheimer’s disease,7~9~1*,13~*5~4~~44-96~50,51.55~63 This has Psychiatry, University Hospital, Rennes). The controls been attributed to loss of cholinergic neurons in the substantia innominata,‘5,34,40,42,47.52~57~6143 which project to the neocortex and, to some extent, to the hippocampus,3,14,31.37,38,56 or to a decrease in their capacity to synthesize acetylcholine.47 Lesions of other cholinergic nuclei (septum, diagonal band of Broca34,42) are also thought to contribute to the decrease in cholinergic enzyme activity in the hippocampus in Alzheimer’s disease.13*25,45,46.51 The present immunocytochemical study was undertaken to obtain a complete description of the cholinergic innervation of the normal human

suffered from chronic non-neurological diseases. The Alzheimer patients were totally dependent on others for the most basic activities of daily living. The diagnosis of Alzheimer’s disease was established on clinical grounds according to DSM III criteria.* The diagnosis was confirmed posr-mortem by determining the density and distribution of senile plaques in the frontal and temporal neocortex. The methods used for staining and quantification are described below. The values are presented in Table 1. All the Alzheimer patients met the criteria of Khatchaturian30 for the density of senile plaques in these regions of the cortex. Age, post-mortem delay and brain weights of the Alzheimer patients were not significantly different from those of controls (Student’s two-tailed r-test). Tissue preparation

AChE, acetylcholinesterase; acetyltransferase.

Abbreviuiions:

ChAT, choline

The brains were hemisected and cut in the frontal plane into 2-cm slabs. Blocks of tissue containing the hippocampus and the temporal lobe were excised at the level of the 701

G. RANSMAYR et al.

702

lateral or superior temporal sulcus. The blocks were fixed in 4% paraformaldehyde/l5% picric acid, as previously described,22 deep-frozen in powdered dry ice, and stored at - 80°C. Serial frontal sections (40 pm) were cut on a sliding microtome along the rostrocaudal axis of the hippocampus from the uncus to the splenium of the corpus callosum. ChAT-immunohistochemistry was performed on sections taken every 2mm. Adjacent sections were stained with Cresyl Violet in order to delimit the subregions of the hippocampus (Fig. 1) and with hematoxyhn-eosin to assure that no ischemic lesions were present. Qualitative analysis was performed on all ChAT-stained sections. Five equidistant sections, at equivalent levels, were selected from each patient for semi-quantitative and quantitative analysis. These sections were counterstained with Thioflavin-S to visualize senile plaquess3 Immunohistochemistry

Immunohistochemistry was performed with a polyclonal antiserum against human ChAT,” contributed by Dr Hersh. lmmunolabelling was revealed with the double-bridge peroxidase-antiperoxidase method described elsewhere.22,5sThe anti-ChAT antiserum was used at a dilution of 1: 200, determined by incubating duplicate sections (from patient No. 1) with 1: 200, 1: 400 and 1:800 dilutions of the serum, under standard experimental conditions. Since identical patterns of immunoreactivity were obtained at all concentrations, the dilution giving the strongest staining (1: 200) was selected. The antiserum labels both cell bodies and nerve fibres, as determined on sections of striatum (from patient No. 1). Absorption of the antiserum with 48pM human placental ChAT (Sigma) eliminated immunostaining of cell bodies and nerve fibres in the striatum and nerve tibres in the hippocampus. One section from each brain was incubated without the primary antiserum to determine whether there was non-specific labelling due to the secondary antibodies. No staining was observed under these conditions. Semi-quantitative estimation of the density of cholineacetylIran&erase-positive jbres and punctate immunoprecipitates

The subregions of the hippocampus were delimited according to Lorente de No,” as illustrated in Fig. 1. Four control subjects (patient Nos 7, 8, 9 and 10) and four patients with Alzheimer’s disease (patient Nos 1, 2, 3 and 5) were studied. The global densities of ChAT-positive fibres and punctate immunoprecipitates were estimated on the five ChAT-stained sections selected from each patient for qualitative and quantitative analysis (see above), as follows. Three microscopic fields (0.36 x 0.25 mm) were selected at random in subregions of the hippocampus (the proximal

and distal fimbria, the alveus at the CA2 and CA1 levels, the stratum pyramidale of CAl, CA2, CA3, CA4 and the subiculum, and the stratum moleculare) on each of the five sections. They were visiuahzed on a monitor (magnification x 800) and densities were scored blind, on a scale of 0 to 4, by two independent investigators (PC. and E.H.) exercised prior to analysis until inter-observer differences were eliminated (inter-class correlation coefficient for 50 ratings: ICC = 0.7914: P c 0.0001~. The scores of the two investieators were averaged for each of the three fields examined per subregion on all five slides from the eight subjects studied. Analysis of variance (two-factor ANOVA) was performed, region by region, on all eight brains, to determine whether there were: (1) significant differences among the three scores obtained per subregion; (2) significant differences among the five slides covering the rostrocaudal extent of the hippocampus. There were no differences either among the three scores per subregion (all F-values i 1.64) or among the different levels of the hippocampus (ail F-values < 1.91), and no interaction among the two factors (F < 0.13). It was therefore possible to calculate the mean density per region per section and, finally, the mean density per region per subject. The mean regional densities for the control subjects and the patients were then compared with Student’s two-tailed t-test. Quantitative evaluation of the density of choline acetyltransferase-positive fibres and punctate structures

Statistical analysis of the semiquantitative data (see above) indicated that the densities of ChAT-like immunoreactive fibres and nerve terminals were homogeneous within the various subregions of the hippocampus, and throughout its rostrocaudal extent. It was possible, then, to undertake a quantitative analysis of cholinergic innervation in the hippocampus of control subjects and patients with Alzheimer’s disease. Four controls and six patients with Alzheimer’s disease were studied. Fibre and terminal densities were quantified as follows. ChAT-immunoreactive fibres were counted (magnification x 2000) in two randomly chosen fields (0.1 x 0.14 mm) in the fibre tracts of the hippocampus (distal fimbria, proximal fimbria and its medial edge, alveus along CA2, and along the proximal and distal parts of CAl), on each of the five sections selected from each patient. The numbers of fibres in the two microscopic fields per region were averaged on each section. These values were then analysed (Kruskal-Wallis ANOVA). region bv region, on the ensemble of the 10 brains to’ de&mine whether there were significant differences in the regional densities among the five slides representing the rostrocaudal extent of the hippocampus. No significant differences were found (all

Table 1. Characteristics of patients Temporal cortex: SP/mm2

Frontal cortex: SP/mm2

980

72 64

66 60

14 I1 9 28

1170 1170 1150 1180

26 40 52 54

12 16 49 43

10 19

1210 1490 1220 940

Post-mortem

Clinical diagnosis

Duration (years)

Age (years)

Sex

2

67 70

m f

Alzheimer* Alzheimer

3 10

3 4 5 6

84 87 80 83

f f m f

Alzheimer Alzheimer Alzheimer Alzheimer

3 15

7 8 9 10

76 75 78 92

f m f f

Control Control Control Control

-

No. I

I

-

Associated pathology Bronchopneumonia Bronchopneumonia Liver cancer Bronchopneumonia

Bronchopneumonia Breast cancer Pulmonar embolism Bronchopneumonia Bladder cancer Pulmonar embolism

delay (h) 4 4

*Familial Alzheimer’s disease. SP: senile plaques (Bodian silver impregnation).

Brain weight (g)

-

0 0

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703

Fig. 1. Subregions of the hippocampal formation. Traced by computer on ChAT-immunostained section (control No. 9). Scale bar = 2 mm. CAl, 2,3,4: Ammon’s horn; GR: granule layer; HIL: hilus (polymorph layer); LM: stratum lacunosum moleculare; MOL: stratum moleculare; SUB: subiculum.

H-values ~3.1). The mean regional fibre densities per square millimetre were then calculated for each subject. The control subjects and patients with Alzheimer’s disease were compared with Student’s two-tailed t-test. Punctate immunoprecipitates and fibre varicosities were counted (magnification x 2000) within randomly chosen fields (0.1 x 0.14 mm) of neuron-containing subdivisions of the hippocampus. A discontinuous grid with 20 openings, 81 pm2 each, was superimposed on the fields; i.e. a total surface area of 1620pm* (20 x 81 pm) distributed over a field of 14,000 hum2(0.1 x 0.14 mm). The values obtained in each of the 20 squares were summed. The procedure was performed four times on the stratum pyramidale of CAI, and twice on the stratum pyramidale of CA4, CA3, CA2, the subiculum, and the stratum moleculare, the polymorph layer and the stratum lacunosum moleculare at the level of CA2 and CA1 (Figs I and 2). Values for fields within a region were averaged. The densities of punctate immunoprecipitates and fibre varicosities per region per section were then analysed by Kruskal-Wallis ANOVA, as for fibre density (se%abovk). No significant differences in the regional densities were found among the five sections representing the rostrocaudal extent of the hippocampus (all H-values ~4.6). The mean regional densities of punctate immunoprecipitate and fibres per square millimetre were then calculated for each subject. The control subjects and patients with

Alzheimer’s disease were compared using Student’s twotailed r-test. Quantification

of senile plaques

To confirm the diagnosis of Alzheimer’s disease, silverimpregnated senile plaques* were counted (magnification x 10) on coronal sections through the frontal cortex (Brodmann area 10) and the temporal cortex (Brodmann area 21). The plaques were counted on a surface of 1 mm2 in the regions of highest density. This procedure was repeated, for each cortex, a minimum of three times per section on three adjacent sections. The mean plaque density per square millimetre was then calculated (Table 1). In the hippocampus, all senile plaques stained with Thioflavin-S and those containing ChAT-immunoreactive fibres or immunoprecipitates were counted (magnification x 25) in the regions of the hippocampus shown in Fig. 1, on all five sections from each patient. The densities per region per section were calculated by dividing the number of senile plaques in each region by the surface area of the region, determined with the Histo 200 image analysis system (Biocorn). The mean regional densities for each subject were then calculated from the five sections representing the rostrocauda1 extent of the hippocampus. Control subjects and patients with Alzheimer’s disease were compared with Student’s two-tailed t-test.

704

G. RANSMAYRet al.

Fig. 2. Layers of the hippocampus at CA2 level. ChATstained section from control No. 7. Scale bar = 200 pm. ALV: alveus; GR: granule layer; HIL: hilus (polymorph layer); hf: hippocampal fissure; LM: stratum lacunosum moleculare; Iv: lateral ventricle; MOL: stratum moleculare; OR: stratum oriens; PYR: stratum pyramidale; RAD: stratum radiatum.

RESULTS Qualitative description of choline acetyltransferaseimmunoreactive jibres and punctate structures in the human hippocampus No ChAT-positive cell bodies were found in any of the sections of the entire hippocampal formation of the 10 brains studied, although they were visible in

the fragment of caudate nucleus present on the tissue sections. Both straight and twisted unbeaded fibres

(diameter 0.8-4 pm) were observed in the hippocampal fibre tracts (fimbria, alveus, alvear path, hippocampal fissure) and in the white matter of the parahippocampal gyrus adjacent to the alvear path (Fig. 3). Punctate immunoprecipitates and fine beaded fibres could be Seen in the neuronal fields of the hippocampus: strata oriens, pyramidale, radiaturn, lacunosum moleculare, moleculare, granulare and polymorph (Fig. 2). The unarborized fibres in the fimbria were scattered in the distal part of the structure, but became tightly aligned just before fanning out into the hippocampus proper. They then coursed along the alveus, decreasing in density towards the angular bundle. Fibres of similar structure and thickness formed a herringbone pattern on each side of the angular bundle, some of which were radially oriented towards CA1 or the subiculum, others towards the temporal neocortex (Figs 3, 4A and B). Sparse unbeaded and relatively straight fibres were visualized entering into the neuronal layers, traversing long distances in various directions. Fibres of similar morphology but finer calibre were observed branching off from these fibres (Fig. 4C). In the neuronal layers, thin beaded fibres (diameter <0.5 pm) forming irregular networks were observed, in particular in the stratum pryamidale of CA 1, CA4 and the subiculum (Fig 4D). Punctate ChAT immunostaining was conspicuous in the stratum pyramidale of CA2 and CA3 (Fig. 4E). The distribution was homogeneous within each part of the structure, but the density differed between the subregions: greater in CA2 than in CA3 and CA4, it decreased gradually from CA2 to the subiculum. Similar or smaller amounts of punctate immunostaining were also visible in the stratum oriens, and still less in the stratum lacunosum moleculare and the stratum radiatum. Occasionally, varicose fibres of medium calibre (0.5-1.5 pm) were observed in the neuronal layers of the hippocampus proper, traversing long distances (Fig. 4F). Dense punctate immunostaining could be observed in the supra- and infra-granular layers of the fascia dentata, bridging the granular cell layer at regular intervals (Fig. 5). There was a relative paucity of fine varicose fibres and punctate immunoprecipitate in the stratum polymorph of the fascia dentata. Frequently, a layer containing a low density of varicose fibres and punctate immunoprecipitates separated the supra-granular layer from a medium dense network of thin, beaded fibres in the outer two thirds of the stratum moleculare. Qualitative comparison of patients disease and control subjects

with Alzheimer’s

ChAT-positive fibres, including the afferent tract in the parahippocampal gyrus, and punctate structures were as intensely stained in hippocampi of patients with Alzheimer’s disease as in controls. Their distribution was similar, but their density appeared to be

Fig. 3. Cholinergic innervation of the hippocampal formation. A. ChAT-immunostained section from control No. 9. Scale bar = 2 mm. B. Schematic representation of the orientation and density of ChAT-positive nerve fibres. AB: angular bundle; FF: fimbria-fornix; CN: caudate nucleus; other abbreviations as in Fig. 2.

Fig. 4. ~o~~o~o~y of CbAT-positive fibres and punctate i~m~no~~~~itates. A. Straight unbeaded tibres in the alveus at CA2 level (control No. IO). Scale bar = 200 gm. B. Twisted fibres in fimbria at CA2 level (control No. IO). Scale bar 2: 100 pm, C. Branched fibres in stratum pyramidale at CA2 level (control No. 10). Scale bar jigIOOgm. D. Network of fine varicose fibres in stratum pyramidale at CA1 level (control No. 7). Scale bar = 100 ym. E. Punctate immunoprecipitate in stratum pyramidale at CA2 ieveI (con&of No. 7). Scale bar = 50 pm. F. Long, medj~m-ca~jbr~ varicose fibres in CA2 (control No. 7). Scale bar = 20Oflm. 706

Hippocampus in Alzheimer’s disease

701

Fig. 5. Punctate immunoprecipitate in the fascia dentata. Arrows indicate ChAT-positive varicose fibres bridging the granule layer (control No. 7). Scale bar = 20 pm. GR: granule layer; iGR: infra-granule layer; sGR: supra-granule layer.

lower in all regions. In certain patients (Nos 1, 5 and 6) the terminals were more or less well preserved in the regions examined, in other patients (Nos 2, 3 and 4), they were to a large extent destroyed. No selective site of degeneration could be detected, but globular immunoprecipitates, thought to represent abnormal

terminals (Fig. 6), twisted fibres and fine fibres which terminated in bulbous, tortuous and enlarged structures, were evident everywhere in the neuronal layers. These changes were also observed, albeit to a minor extent, in controls. Except for the deformed terminals, there were no consistent qualitative abnormalities in the fibre tracts of the hippocampal formation or in the cholinergic pathway approaching the hippocampus from the ventrolateral direction, where fibre density was also diminished in Alzheimer’s disease. Examples of the fimbria and the stratum pyramidale of CA2 and CA1 are shown in Fig, 7.

Semi-quantitative analysis of cholinergic innervation in control and Alzheimer ‘s disease subjects

Semi-quantitative estimates of the densities of ChAT-positive fibres and punctate structures in subregions of the hippocampus of four control subjects and four patients with Alzheimer’s disease are summarized in Table 2. A significant loss of ChAT-immunoreactive fibres and terminals was observed in most of the regions examined. In the other regions (proximal fimbria, alveus at CA1 level, stratum pyramidale of CA3 and stratum moleculare), the differences approaches significance (P = 0.07, P = 0.10, P = 0.08, P = 0.19, respectively). Quantitative analysis of cholinergic innervation in control and Alzheimer’s disease subjects Fig. 6. Abnormal &AT-positive fibre in bilus. Section from patient No. 5. Scale bar = 200 pm.

Quantitative evaluation of the densities of ChATpositive fibres and punctate structures in subregions

708

CL RANSMAYRet al.

Table 2. Density of choline acetyltransferase-positive nerve fibres and punctuate immunoprecipitates (semi-quantitative data) Control

Alzheimer

Fibres Distal fimbria Proximal fimbria Alveus (CA2) Alveus (CAI)

2.3 f 0.2 2.7 k 0.3 1.5 *to.1 0.8 f 0.2

1.5 f 0.2* 1.7kO.2 0.5 * o.t** 0.4kO.l

Punctate immunoprecipitate Stratum pyramidale Subiculum CA1 CA2 CA3 CA4 Stratum moleculare

1.7 f 0.2 2.3 + 0.2 2.9 + 0.3 2.4kO.l 2.5 f 0.3 1.8 & 0.3

0.8 + 0.2* 1.3 * 0.2* 1.7 + 0.3’ 1.7kO.2 1.2 + 0.4* 1.0 * 0.3

Fibres/mm2 Control Alzheimer Distal fimbria Proximal fimbria Alveus (CA2) Alveus (CAl)

59O+56 873 + 109 324 f 44 302 &-28

412 f 560 f 174 f 202 f

32* 51* 23* 19*

Values represent mean f S.E.M. *P c 0.05.

the density of immunopreciptiates cantly in all structures (2846%).

decreased signifi-

Quantitative analysis of senile plaques in hippocampus of controls and Alzheimer patients

Density scored 0 to 4. Values represent mean + S.E.M. *P < 0.05, **p < 0.01.

of the hippocampus patients

Table 3. Density of choline acetyltransferasepositive nerve fibres (quantitative dam)

of four control subjects and six with Alzheimer’s disease are summarized in

Tables 3 and 4. Fibre density (Table 3) was greatest in the proximal fimbria, followed in order by the distal fimbria and the alveus. The loss was in general 3&35%, but reached 50% in the alveus at the CA2 level. ChAT-positive immunoprecipitate (Table 4) increased in density in control subjects from the subiculum to CA2, then decreased slightly. Lower densities were observed in the stratum polymorph and the stratum moleculare. In Alzheimer patients,

the

Thioflavin-S-stained senile plaques were numerous in the hippocampal formation of Alzheimer patients (Table 5). A few were also found in control subjects (Table 5). Plaque density varied from subject to subject and region to region. Highest mean densities were found in the outer two-thirds of the stratum moleculare of the fascia dentata (in the lower onethird almost no plaques were visible) and the stratum pyramidale of the subiculum and CAl. The majority of Thioflavin-S-stained senile plaques in Alzheimer patients and practically all Thioflavin-S-stained plaques in controls contained either normal or distorted, irregular ChAT-positive fibres or punctate or bulbous deposits (Fig. 8). The percentage of Thioflavin-S-stained senile plaques containing

Table 4. Density of choline acetyltransferase-positive (quantitative data)

punctate immunoprecipitate

Punctate immunoprecipitate x 10-5/mm2 Alzheimer Controls Stratum pyramidale Subiculum CA1 CA2 CA3 CA4 Stratum lacunosum moleculare Stratum moleculare Stratum polymorph

3.6 f 0.2 5.0 * 0.2 7.7 + 0.4 6.9 + 0.3 6.8 f 0.4 2.5kO.l 4.2 k 0.2 4.4 f 0.3

2.1 + 3.1 * 5.3 + 4.4 + 3.9 rt 1.8 f. 2.8 f 3.1 f

0.2’2 0.4** 0.7* 0.71 0.6** O.l* 0.4* 0.1**

Values represent mean + S.E.M. *P < 0.05, **P < 0.01. Table 5. Density of senile plaques

Stratum pyramidale Subiculum CA1 CA2 CA3 CA2 Stratum lacunosum moleculare Stratum moleculare Stratum polymorph

SP(THI0) x lo-‘/mm’ Alzheimer Control

SP (ChAT) x lo-‘/mm* Alzheimer Control

0.1 kO.1 0.1 k 0.2 0 0.2 * 0.2 0.1 + 0.1 0.1 f 0.2 0.3 + 0.3 0.1 + 0.2

0.1 * 0.1 0.1 * 0.2 0 0.2 * 0.2 0.1 & 0.1 0.1 +0.2 0.3 & 0.3 0.1 + 0.2

7.0 f 2.3+ 6.1 + 0.9* 1.5 f 0.3’ 1.7 * 0.7* 3.3 + 1.4* 3.6 f 0.9* 10.9 f 4.5* 4.3 &-1.5+

4.7 + 4.3 + 1.3 + 1.4 + 2.8 + 2.3 + 9.7 + 2.2 f

1.7* 0.6’ 0.3+ 0.7. 1.2* 0.6* 4.0* 0.9.

SP (ChAT) SP (THIO) Alzheimer Control 1 1 1 1 1

1 1 1

0.67 0.70 0.87 0.82 0.85 0.65 0.88 0.51

SP(THI0): senile plaques stained with Thioflavin-S; SP(ChAT): senile plaques containing ChAT-like immunoreactivity. Values represent mean k S.E.M. *P c 0.001.

Hippocampus in Alzheimer’s disease

Fig. 7. Cl&T-positive fibres and punctate immunoprecipitate in control subjects and patients with Alzheimer’s disease. Scale bar = 1OOpm. A and B. Proximal timbria adjacent to alveus at CA3 level (A-control No. 7; B-Alzheimer patient No. 2). C and D. Stratum pyramidale at CA2 level (C--control No. 7; D-Alzheimer patient No. 1). E and F. Stratum pyramidale at CA1 level (E-control No. 7; F-Alzheimer patient No. 1).

ChAT-like immunoreactivity also varied among patients and regions. The highest percentages were found in the stratum moleculare (outer two-thirds), the stratum pyramidale of CA2, CA4, CA3 and CA 1, followed by the subiculum, the stratum lacunosum moleculare and the stratum polymorph.

Numerous neurofibrillary tangles were also present in Alzheimer brains and to a lesser extent in controls. Highest densities were observed in the stratum pyramidale of CA1 and the subiculum. None was ChAT-positive; quantification, therefore, was not undertaken.

710

G.

RANSMAYR et

Correlations No correlations

were found between the density of ChAT-positive fibres, terminals or senile plaques in the hippocampus and age, disease duration, postmortem delay or senile plaque density in the temporal and frontal cortex. A trend towards a linear correlation (r = 0.54; P = 0.14) was observed between the mean density of ChAT-positive punctate structures and the percentage of Thioflavin-S-stained plaques

Fig. 8. Senile plaque. A. ChAT-positive nerve fibre in a senile plaque in the stratum polymorph (Alzheimer patient No. 1). Scale bar = 20 pm. B. The same plaque stained with Thioflavin-S.

al.

containing ChAT-like immunoreactivity various subregions of the hippocampus.

in

the

DLSCUSSION Distribution of nerve jibres and terminals containing choline acetyltransferase-like immunoreactivity in the hippocampus of control subjects

In the present study of the human hippocampus, the distribution of cholinergic fibres and punctate structures, taken to be nerve terminals,4~s9was similar to that described in rats,26@ rabbits,*’ guinea-pigs” and non-human primates. 3’All neuronal layers in the hippocampus were innervated by cholinergic fibres. The high density of cholinergic fibres in the fimbria and the distribution of the fibres in subregions of the hippocampus suggest that, analogous to other animal species, the major cholinergic input to the human hippocampus enters the structure via the fimbria-fornix and most likely originates, for the most part, in the vertical limb nucleus of the diagonal band of Broca (Ch2) and in the medial septal nucleus (Chl).‘~6,3’*32~35.37,)8,43.60 The pattern formed by cholinergic fibres along the alvear path and in the white matter of the parahippocampal gyrus suggests that the human hippocampus also receives a minor input ventrally via the white matter of the temporal lobe, originating presumably in the diagonal band of Broca and the nucleus basalis of Meynert, as observed in rodents” and non-human primates,-” respectively. The presence of ChAT-positive varicosities, punctate or globular immunoprecipitates and both normal and abnormal cholinergic fibres in Thioflavin-Sstained senile plaques suggests that the cholinergic innervation of the hippocampus suffers some degenerative changes during normal ageing. That these ChAT-positive elements belong to nerve processes has been previously demonstrated.” Since practically all of senile plaques in the normal brains involved choline&c fibres, unlike in the pathological cases discussed below, choline@ neurons may be particularly, or precociously, prone to degeneration. No ChAT-immunoreactive perikarya could be seen in the human hippocampus, in the present study, in contrast to immunocytochemical observations in the rat.‘6*26@Technical considerations, such as tissue fixation, antibody concentrations or incubation conditions, might explain this discrepancy. This seems unlikely, however, since ChAT-immunoreactive cell bodies were easily visualized in the striatum in control sections (see Experimental Procedures) and on the portions of caudate nucleus visible on the hippocampal sections used in this study (Fig. 3). This study is, to our knowledge, the first performed on human hippocampus in which choline@ innervation was investigated with a selective antiserum against ChAT, a marker specific for cholinergic neurons, unlike AChE histochemistry. A certain number of differences in the staining patterns of putative choline@ innervation in the human

Hippocampus in Alzheimer’s disease

hippocampus obtained with the two techniques should be noted. No cell bodies were stained in the human hippocampal formation with the anti-&AT antiserum, whereas AChE-positive perikarya have The latter may be non-cholinbeen reported. 23,36,46 ergic neurons which contain the enzyme,24 but it cannot be excluded that the neurons either contain more AChE than ChAT facilitating visualization, or that AChE histochemistry, while less specific than ChAT immunocytochemistry, is a more Sensitive method of detection. Secondly, the relative densities of nerve terminals in the subregions of the hippocampus differed: the highest density of ChAT-positive terminals was found in the stratum pyramidale and juxtapyramidale zone of the stratum oriens; AChEpositive neuropil predominates in the strata oriens and the juxtapyramidal zone of the stratum radiatum, and is lower in the stratum pyramidale.36l46 Thirdly, ChAT-immunoreactive neurofibrillary tangles were not observed in the subjects studied here, although AChE-containing tangles have been consistently demonstrated.39

Choline acetyltransferase hippocampus of patients

immunohistochemistry in the with Alzheimer’s disease

If ChAT-positive immunostaining indeed reflects the distribution of ChAT present in the tissue, the results suggest that there may be significant differences between the choline@ innervation of the hippocampus in controls and patients with Alzheimer’s disease. The densities of both the fibres and nerve terminals were significantly decreased in Alzheimer patients compared to controls in all regions of the hippocampal formation when evaluated quantitatively, and in most regions when estimated by the semi-quantitative method. Both dorsal and ventral input was affected. There were marked differences, however, in the extent of the decrease among individual subjects. The values for controls and patients with Alzheimer’s disease overlapped. A number of factors could contribute to the intersubject variability: endogenous differences, genetic or developmental, governing the original density of the cholinergic innervation in the individuals; pre- and post-mortem conditions affecting the activity of the marker enzymes,45,49,51experimental variables (tissue fixation and conservation, irreducible interexperimental variations). In spite of these sources of scatter, and the small number of subjects in the control and Alzheimer groups, the decrease in cholinergic innervation throughout the hippocampus of the Alzheimer patients was significant, and may therefore be suspected to result from the disease. In several studies on patients with Alzheimer’s disease,‘8~27~”enhanced AChE staining was observed in the neuropil in the outer two-thirds of the stratum moleculare, and was interpreted as evidence of compensatory sprouting of septohippocampal cholinergic neurons. This increase was not observed in the

711

present study, where, on the contrary, ChAT-like immunoreactivity decreased in this structure. It cannot be excluded, however, that sprouting could not be detected with the methodology used. The decrease in ChAT-immunoreactive nerve fibres and terminals in the hippocampus of Alzheimer patients is in agreement with assays of ChAT activbut the percentage decrease observed in ity, 72’3~25344*50~5’ these studies (40-87%) is generally greater than that reported here. The reason for this difference is not clear. In the present study, even the patient with relatively early onset familial Alzheimer’s disease, where neuronal lesions are reputed to be severe,29 had substantial ChAT-like immunoreactivity. The specificity of the anti-&AT antiserum for the enzyme has been verified (see Experimental Procedures), so it seems unlikely that non-cholinergic nerve fibres have been labelled. It is possible, however, that an immunologically recognizable but inactive, or less active, form of the enzyme is present in the cholinergic neurons remaining in Alzheimer patients, or that preserved cholinergic structures in Alzheimer’s disease contain less ChAT than similar structures in controls. Molecular biological studies on ChAT expression in this disease will eventually provide the answer to this enigma. Is there a relationship between choline& innervation of the hippocampus and the presence of senile plaques? The number of plaques stained with Thioflavin-S in the hippocampus of individual patients paralleled the number of Bodian-stained senile plaques counted in the neocortex for diagnostic purposes (Table l), suggesting that the neocortex and hippocampus are not dissociated with respect to the pathological process. Both Thioflavin-S-stained and ChAT-positive senile plaques were distributed throughout the hippocampus, in a pattern similar to that described by others. 7*21,54 The patterns of both Thioflavin-S- and ChAT-stained senile plaques were heterogeneous, however, whereas the loss of cholinergic terminals in the patients was quite homogeneous. There is then no obvious relationship between the degeneration of ChAT-positive fibres and the presence of senile plaques, even those, which are ChAT-positive. However, there is a tendency towards a significant correlation between the percentage of senile plaques in a given region that contains ChATpositive nerve fibres or terminals and the density of cholinergic fibres in that region. The correlation would probably become significant if a greater number of patients was studied. A significant correlation between the percentage of ChAT-positive senile plaques and the density of ChAT-positive nerve fibres in the neocortex, the amygdala and the hippocampus of patients with Alzheimer’s disease, has previously been reported,5 but subregions of the hippocampus were not examined in this study. It would seem, then, that if not all senile plaques can be attributed to a pathology affecting cholinergic neurons, there is a relationship in a certain number of cases.

712

G. RANSMAYR et al. CONCLUSION

This immunocytochemical study of the human hippocampus with an antiserum against ChAT indicates that a major afferent cholinergic pathway enters the structure dorsally via the fimbria-fornix, a minor afference entering from the temporal lobe following the alvear path. No ChAT-containing intrinsic neurons were detected. The cholinergic innervation suffers some degenerative change in normal aged subjects, but decreases

considerably

in density in Patients

with

Alzheimer’s disease. The decrease differs somewhat

among the subregions of the hippocampus, but is homogeneously distributed within each subregion, and throughout the rostrocaudal extent of the structure. Compensatory sprouting in reaction to denervation was not detected. Acknowledgements-The authors are grateful for the financial support provided in France by the Caisse Nationale d’Assurance Maladie des Travailleurs Salaries (grant 126/87), in Austria by the Austrian Fund for the Advancement of Scientific Research (grant J0236M), and in the United States by the National Institute of Health (grant AG 05893).

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