0306-4522/92$5.00+ 0.00 PergamonPress plc © 1992IBRO
Neuroscience Vol. 47, No. 4, pp. 843-851, 1992 Printed in Great Britain
ALZHEIMER'S DISEASE: IS THE DECREASE OF THE CHOLINERGIC INNERVATION OF THE HIPPOCAMPUS RELATED TO INTRINSIC HIPPOCAMPAL PATHOLOGY? G. RANSMAYR,*I"P. CERVERA,~E. C. HIRSCH,~W. BERGER,t W. FISCHER§and Y. AGID~ 1"Universit/itsklinikfiir Neurologie, Anichstra~ 35, 6020 Innsbruck, Austria $INSERM U289, Hrpital de la Sall~trirre, 47 Bd. de l'H6pital, 75013 Paris, France §Universit//ts-Institut fiir Physiologie, Fritz-Pregl-Stral3e3, 6020 Innsbruck, Austria Abstract--Consistent findings in the hippocampi of patients with Alzheimer's disease are the presence of neurofibrillary tangles in pyramidal neurons and the loss of choline acetyltransferase activity due to degeneration of hippocampal cholinergic terminals. The present study sought to clarify, in the brains of five patients with Alzheimer's disease and four controls, whether the loss of cholinergic terminals in the hippocampal stratum pyramidale in Alzheimer's disease is related to degenerativechanges in hippocampal pyramidal cells. A polyclonal antibody to human choline acetyltransferase was employed to visualize immunohistochemically cholinergic terminals. Hippocampal neurons were stained with Cresyl Violet, neurofibrillary tangles with thioflavin S and a monoclonal antibody against phosphorylated neurofilament (RT97). Quantification of the stained structures was performed in CA4, CA1 and the subiculum, on five sections selected from the entire anteroposterior extent of each hippocampus. In the group of Alzheimer patients, the densities of cholinergic terminals were homogeneously diminished in the three hippocampal subregions in comparison with the controls (32-33%). In contrast, a significantloss of pyramidal neurons was found only in CA1, and the density of neurofibrillary tangles was markedly increased only in CA1 and the subiculum in Alzheimer's disease. These findings suggest that there is no relationship between the loss of cholinergic terminals and the degeneration of pyramidal cells in the hippocampus of patients with Alzheimer's disease.
Previous immunohistochemical studies in rodents employing antibodies to choline acetyltransferase (CHAT) demonstrated that the hippocampus contains fine, beaded, ChAT-immunoreactive, and, thus, cholinergic fibers and punctate structures exhibiting the electron microscopic characteristics of nerve terminals establishing synaptic contact with hippocampal pyramidal neurons. 14'1s'45 The majority of these cholinergic terminals in the hippocampus are thought to originate from cholinergic nuclei in the basal forebrain (diagonal band of Broca, medial septal nucleus and nucleus basalis of Meynert). 22'24'29 Two cholinergic pathways from the basal forebrain region to the hippocampus were identified in animals, one in the funbria-fornix system, and a second in the white matter of the temporal lobe. 22,24,29The anatomy of the human hippocampal cholinergic system is similar to that in rodents except for the absence of detectable intrinsic ChAT-immunoreactive perikarya. 16,ls'2s'37,39,a3,45The presence of gross, unbeaded ChAT-immunoreactive fibers in the fimbria and the parahippocampal gyrus and the absence of CHATimmunoreactive intrinsic perikarya suggest that the cholinergic innervation of the human hippocampus stems from extrinsic cholinergic perikarya, presumably located in the basal forebrain region. 27,33,39 *To whom correspondence should be addressed. Abbreviation: CHAT, choline acetyltransferase.
In Alzheimer's disease, both the cholinergic nuclei in the basal forebrain region27,~ and the cholinergic terminals in the hippocampal region ~1'~7'34'36'37'39-4' undergo degeneration. The hippocampi of patients with Alzheimer's disease also contain numerous senile plaques. Moreover, hippocampal pyramidal neurons are affected in Alzheimer's disease; high densities of neurofibrillary tangles and a loss of neurons are found in the hippocampal stratum pyramidale.5,6.19,3]'3s A previous study showed that the density of senile plaques in the hippocampus of patients with Alzheimer's disease is not related to the loss of cholinergic terminalsfl Whether or not the degeneration of hippocampal pyramidal neurons is related to the loss of cholinergic terminals is so far unclarified. We compared, therefore, the decrease of cholinergic terminals in the stratum pyramidale to the loss of pyramidal cells and the density of neurofibrillary tangles in the hippocampus of patients with Alzheimer's disease.
EXPERIMENTAL PROCEDURES
Characteristics of the patients and controls
The brains of four control subjects who had suffered from various non-neurological diseases and five patients with Alzheimer's disease exempt from vascular disease, brain tumor, infections and other neuropsychiatric disorders were studied (Table 1). Clinical diagnosis of Alzheimer's disease was based on the criteria of the DSM Ill. t The Alzheimer
843
G. RANSMAYR et al.
844
Table 1. Clinical and pathological characteristics of the patients
No. 1 2 3 4 5 6 7 8 9
Age (years)
Sex
Clinical diagnosis
76 75 78 92 67 70 87 80 83
f m f f m f f m f
control control control control Alzheimer Alzheimer Alzheimer Alzheimer Alzheimer
Disease duration (years) .... 3 3 15 5 1
Associated pathology Pulmonary embolism Bronchopneumonia Bladder cancer Pulmonary embolism Bronchopneumonia Bronchopneumonia Bronchopneumonia breast cancer
Post mortem delay (h)
Brain weight (g)
10 19 5 7 4 4 11 9 28
1210 1490 1220 940 980 1170 1150 1180
Temporal Frontal cortex cortex (SP/mm 2) (SP/mm 2) 1 0 1 0 72 64 40 52 54
3 I 0 0 66 60 16 49 43
For statistical evaluation see Experimental Procedures (Characteristics of the patients and controls). SP, senile plaques. patients had been totally dependent on others for the most basic activities of living. Ages and post mortem delays were not different in the Alzheimer and control groups (P = 0.807 and 0.806; Mann-Whitney U-test). The weights of the brains were significantly higher in the controls compared with the patients with Alzheimer's disease (P =0.029, Mann-Whitney U-test). Post mortem neuropathological examination using the Bodian silver impregnation technique demonstrated high densities o f neuritic plaques (Table 1) and neurofibrillary tangles (not shown) in the frontal and temporal neocortex (Brodmann areas 10 and 21) and the hippocampal formation, meeting the histological criteria for Alzheimer's disease. 2t Histopathological examination of the frontal and temporal cortex and the hippocampus of the controls revealed only sparse neuritic plaques (Table 1) and low densities of neurofibrillary tangles (not shown).
Tissue preparation and immunohistochemistrv of the hippocampus The brains were dissected, fixed, cryoprotected and deep-frozen, as described elsewhere. 39 The entire anteroposterior extents of the hippocampi were cut serially on a sliding microtome into 40-#m-thick frontal sections. ChAT immunohistochemistry was performed on sections taken every 2 mm with a polyclonal antiserum directed to human CHAT. 9 The antiserum to ChAT was well characterized and its specificity had been verified in previous studies. 4,7,9,23,39 The immunohistochemical procedures are described elsewhere) s'44 Sections adjacent to the ChAT-immunostained sections were stained with hematoxylin-eosin to exclude vascular lesions. Bodian's silver-impregnation technique was employed to visualize neuritic plaques and neurofibrillary tangles. Sections at a distance of 80/~m from those selected for ChAT immunohistochemistry were treated with Cresyl Violet to visualize neuronal perikarya. To demonstrate neurofibrillary tangles and amyloid plaques together with cholinergic structures, the ChAT-immunostained sections were counterstained with thioflavin S.42 Sections at a distance of 6 0 - 3 2 0 # m from the ChAT-immunostained sections were treated with a mouse monoclonal antibody against phosphorylated neurofilament (RT97). z3° The immunohistochemical procedures, including the dilution of the primary antibody, were identical to those described for ChAT immunohistochemistry except for the use of a goat anti-mouse secondary antibody (dilution 1:10) and a mouse peroxidase-antiperoxidase complex (dilution 1: 500). Histological structures quantified and regions analysed The entire anteroposterior extent of each hippocampus was divided into five segments of equal length. From each segment, a series of sections was selected to quantify in the stratum pyramidale ChAT-immunostained structures. RT97 and thioflavin S-stained neurofibrillary tangles, and Cresyl Violet-stained cell bodies.
Quantitative analyses of ChAT-immunoreactive structures, neurofibrillary tangles and neurons were performed in the CA4 and CA1 sectors and the subiculum. CA4, CAI and the subiculum were distinguished on Cresyl Violetstained sections according to criteria described elsewhere ~6.25 (Fig. 1). In brief, the extension of the stratum pyramidale into the area surrounded by the stratum granulare was defined as the CA4 sector. The transition from CA2 to CAI is characterized by a gradual widening of the stratum pyramidale. Moreover, in CAI the density o f pyramidal neurons is lower than in CA2, the cell bodies are larger ("giant neurons"), and the stratum pyramidale is divided into two main layers of neurons. The transition from the CA1 sector to the subiculum is characterized by a loss of the stratum radiatum of CA1, a gradual narrowing of the layer of hippocampal pyramidal cells and a widening of a subjacent layer of darker stained and less regularly shaped pyramidal neurons designated as subicular pyramids. 25 "'Clouds" of moderately stained and densely packed perikarya overlying the subicular pyramids mark the transition from the subiculum to the presubiculum.
Quantification of choline acetyltransferase-positive punctate immunoprecipitates The preponderant ChAT-immunostained structures in the stratum pyramidale were fine, beaded fibers and punctate immunoprecipitates characterized elsewhere as fiber varicosities and axon segments of cholinergic terminals t4:s'45 (Fig. 2A, C). The density of punctate ChAT-immunoprecipitates was taken as an index of the density of cholinergic terminals, t4:sm In order to verify whether ChAT-immunostained punctates were homogeneously distributed within the hippocampus and sampling methods could be used, one ChAT-immunostained section was selected from the central part of each hippocampus and analysed as follows. In CA4, CA1 and the subiculum, the longitudinal extent of the stratum pyramidale was divided into five parallel layers of equal width oriented in parallel with the outer circumference of the hippocampus. The five layers were spaced into 0.14 x 0.1 mm microscopic fields arranged consecutively at 0.14 mm intervals along the entire longitudinal extents of the three subregions. The fields were visualized at × 2000 magnification on a 24 × 20 cm videoscreen covered with two 18 x 18 mm square openings separated by 10cm (corresponding to two 9 x 9 # m microscopic fields separated by 50/~m). Quantification of ChAT-immunoreactive punctate structures was conducted on the 18 x 18 mm squares on the videoscreen. To analyse whether there were differences in the densities of ChAT-positive punctate immunoprecipitates between the five layers, one-way analysis of variance was performed on each of the nine selected sections separately in the three regions. No statistical differences were found between the five layers on any section in any region (F =2.116 to 0.19; P =0.117 to 0.941). Therefore. on the remaining sections of the nine brains examined,
Cholinergic system in Alzheimer hippocampi
845
Fig. 1. (A) Representative Cresyl Violet-stained frontal section (40/~m thick) through the mid-part of the anteroposterior extent of the hippocampus of control brain 4. The stratum pyramidale of CA4, CA1 and the subiculum is outlined according to the methods described in the text. PRE, presubiculum; SUB, subieulum. (B) Section at a distance of 80/am along the anteroposterior extent of the hippocampus from the section shown in A labeled with a polyclonal antibody specific for human placental CHAT.
the densities of ChAT-positive punctate immunoprecipitates were quantified only in the layer in the midline of the stratum pyranaidale with an otherwise identical method. Finally, the mean densities of ChAT-immunostained punctates were calculated for each subregion of each section.
Quantification of thioflavin S- and RT97-positive neurofibrillary tangles The thioflavin S-positive neurofibrillary tangles (Fig. 2E) were visualized under UV fight (Olympus, BH2-DMB filter block, green filter). Quantification of RT97 (Fig. 2F) and thioflavin S-stained tangles was performed on the five selected sections for each staining within the entire surface areas of CA4, CAI and the subiculum. The surface areas of the stratum pyramidale of the subregions were determined with a semiautomatic image analysing system (Histo 2000, Biocom, France). To obtain surface densities of neurofibrillary tangles for each subregion on each section, the number of tangles per subregion was divided by the surface areas.
Quantification of Cresyl Violet (Nissl)-stained neurons Staining with Cresyl Violet revealed, in the hippocampal pyramidal layers, triangular, ovoid or polygonal, blue perikarya (Fig. 2B, D). Quantification was performed with a x 40 objective (= x 400 magnification) in 0.25 x 0.25 mm fields spaced consecutively at 0.25 nun intervals along the entire longitudinal extents of the selected subregions in two layers centered in the inner and outer halves of the radial diameter of the stratum pyramidale. Only neurons containing a dark blue nucleolus were counted. Finally, the mean densities of neurons per mm 2 were calculated for each subregion of each section.
Statistical analysis The Friedman test was used to analyse the controls and the Alzheimer group separately, whether there were differences in the densities of ChAT-positive punctate immunoprecipitates, thiotlavin S- and RT97-positive tangles and Cresyl Violet-stained neurons in CA4, CA1 and the subiculum between the five levels of the anteroposterior extent of the hippocampus. No differences were found between the five levels in the two groups (Chi- squared scores ranging
from 1.2 to 8.7; P from 0.878 to 0.074). Therefore, the subregional density scores of the five sections of each hippocampus were averaged and the resultant mean densities of ChAT-positive punctate precipitates, neurofibrillary tangles and neurons for the Alzheimer and the control brains compared with the Mann-Whitney U-test. In addition, Kruskal-Wallis ANOVA and Mann-Whitney U-test were applied to compare, in the Alzheimer and the control groups separately, CA4, CA1 and the subiculum as to differences in the mean densities of the structures quantified with the three subregions. RESULTS
Choline acetyltransferase-positive punctate immunoprecipitates in the hippocampus of controls and patients with Alzheimer's disease In the controls, the densities of ChAT-positive punctate immunoprecipitates were significantly higher in CA4 than in CA1 and the subiculum, and in CA1 compared with the subiculum (Table 2). In the Alzheimer group, significantly higher densities of ChAT-positive punctate immunoprecipitates were found in CA4 and CA1 compared with the subiculum. The densities of ChAT-positive punctate immunoprecipitates in CA4, CA1 and the subiculum were significantly diminished in the Alzheimer group as compared to the controls (Table 2; Fig. 2A, C). There was a mean 32-33% loss of ChAT-positive terminals in the three subregions in the Alzheimer group. The decrease of ChAT-immunoreactive punctate structures in the Alzheimer group varied from patient to patient (almost no loss in patient 9 and marked loss in patient 6). In the individual patients, however, no major differences in the decrease of ChAT-positive punctate immunoprecipitate were found between the three subregions.
846
G. RANSMAYRet al.
Fig. 2. Photomicrographs taken from the CA1 sector, stratum pyramidale, of the mid-part of the anteroposterior extent of the hippocampus of control brain number 1 (A, B) and Alzheimer brain 7 (C-F). Scale bar = 50/zm. (A) ChAT immunohistochemistry revealing in the normal control brain networks of fine, beaded fibers and dense punctate immunoprecipitates characterized elsewhere as cholinergic terminals (see text). (B) Cresyl Violet-staining of a control brain section revealing hippocampal pyramidal neurons. The arrow points to a nucleolus. (C) ChAT immunohistochemistry in Alzheimer's disease showing remnants of punctate immunoprecipitates and fine and beaded fibers with partly globular enlargements. These changes of cholinergle terminals are found characteristically in Alzheimer's disease (arrows). (D) Cresyl Violet-stained pyramidal neurons in an Alzheimer brain. The arrow points to a nucleolus. (E) Dark-field photomicrograph (UV light, BH2-DMB filter block, Olympus) of the microscopic field shown in bright-field in C, counterstained with thioflavin S to demonstrate neurofibrillary tangles (long arrows). The short arrow points to the ChAT-immunoreactive beaded fiber visualized in C. (F) Neurofibrillary tangles visualized immunohistochemically with a monoclonal antibody to phosphorylated neurofilament (RT97).
Neurofibrillary tangles in the hippocampus of controls and patients with Alzheimer's disease In the controls, the densities of thioflavin S-stained neurofibrillary tangles (Fig. 2E) did not differ between CA4, CA1 and subiculum (Table 3). In the Alzheimer group, density of thioflavin S-stained neurofibrillary tangles was higher in CAI compared
to CA4, whereas no differences were found between CA1 and the subiculum, and CA4 and the subiculum. In the three regions analysed the densities of thioflavin S-stained tangles were significantly higher in the brains of patients with Alzheimer's disease than in controls (Table 3). The densities of thioflavin S-stained neurofibrillary tangles in CA4 and CA1 were higher in brains 6 and 7 than in 5, 8 and 9.
847
Cholinerglc system in Alzheimer hippoeampi Table 2. Density of choline acetyltransferase-immunoreaetive punetate precipitates (eholinergic terminals) in the human hippocampal stratum pyramidale Brain number
Clinical diagnosis
CA4
CA 1
Subiculum
1 2 3 4 Mean
control control control control
52.7 __+5.7 45.5 + 5.5 47.3 _ 1.2 52.4 _ 6.7 49.5 + 1.8
41.5 -I- 4.2 40.7 _ 2.9 32,4 + 2.4 42,5 + 2.4 39.3 _ 2.4
28.9 -I- 4.3 25.8 __+3.0 22.9 _+ !.9 33.6 __+3.1 27.9 __+2.3
5 6 7 8 9 Meant
Alzheimer Alzheimer Alzheimer Alzheimer Alzheimer
26.2 __+1.9 20.7+2.1 28.6 + 3.3 39.1 __+1.6 50.8 _ 2.1 33.1 __+5.3*
27.6 __+2.8 19.2+ 1.7 24.6 + 1.6 31.8 __+1.8 29.1 _ 3.3 26.6 _ 2.5*
17.3 __+1.4 13.4+ 1.6 19.2 + 2.6 22.9_ 1.6 20.0 __+2.1 18.6 -+ 1.6"
Results are expressed as means + S.E.M. of 103 ChAT-immunoreactive punctate precipitates per mm 2 of five sections of the individual brains (see Experimental Procedures). Statistical analysis (only significant differences are indicated): interreglonal differences of the means of the controls and the Alzheimer group (Kruskal-Wallis ANOVA and Mann-Whitney U-test): controls, H = 9.27; CA4/CAI*, CA1/Sub*, CA4/Sub*; Alzheimer's disease, H = 7.31; CA1/Sub*, CA4/Sub*. tComparison of the means of CA4, CA1 and the subiculum of the Alzheimer group with those of the controls (Mann-Whitney U-test). *P
Neuroscience Vol. 47, No. 4, pp. 843-851, 1992 Printed in Great Britain
ALZHEIMER'S DISEASE: IS THE DECREASE OF THE CHOLINERGIC INNERVATION OF THE HIPPOCAMPUS RELATED TO INTRINSIC HIPPOCAMPAL PATHOLOGY? G. RANSMAYR,*I"P. CERVERA,~E. C. HIRSCH,~W. BERGER,t W. FISCHER§and Y. AGID~ 1"Universit/itsklinikfiir Neurologie, Anichstra~ 35, 6020 Innsbruck, Austria $INSERM U289, Hrpital de la Sall~trirre, 47 Bd. de l'H6pital, 75013 Paris, France §Universit//ts-Institut fiir Physiologie, Fritz-Pregl-Stral3e3, 6020 Innsbruck, Austria Abstract--Consistent findings in the hippocampi of patients with Alzheimer's disease are the presence of neurofibrillary tangles in pyramidal neurons and the loss of choline acetyltransferase activity due to degeneration of hippocampal cholinergic terminals. The present study sought to clarify, in the brains of five patients with Alzheimer's disease and four controls, whether the loss of cholinergic terminals in the hippocampal stratum pyramidale in Alzheimer's disease is related to degenerativechanges in hippocampal pyramidal cells. A polyclonal antibody to human choline acetyltransferase was employed to visualize immunohistochemically cholinergic terminals. Hippocampal neurons were stained with Cresyl Violet, neurofibrillary tangles with thioflavin S and a monoclonal antibody against phosphorylated neurofilament (RT97). Quantification of the stained structures was performed in CA4, CA1 and the subiculum, on five sections selected from the entire anteroposterior extent of each hippocampus. In the group of Alzheimer patients, the densities of cholinergic terminals were homogeneously diminished in the three hippocampal subregions in comparison with the controls (32-33%). In contrast, a significantloss of pyramidal neurons was found only in CA1, and the density of neurofibrillary tangles was markedly increased only in CA1 and the subiculum in Alzheimer's disease. These findings suggest that there is no relationship between the loss of cholinergic terminals and the degeneration of pyramidal cells in the hippocampus of patients with Alzheimer's disease.
Previous immunohistochemical studies in rodents employing antibodies to choline acetyltransferase (CHAT) demonstrated that the hippocampus contains fine, beaded, ChAT-immunoreactive, and, thus, cholinergic fibers and punctate structures exhibiting the electron microscopic characteristics of nerve terminals establishing synaptic contact with hippocampal pyramidal neurons. 14'1s'45 The majority of these cholinergic terminals in the hippocampus are thought to originate from cholinergic nuclei in the basal forebrain (diagonal band of Broca, medial septal nucleus and nucleus basalis of Meynert). 22'24'29 Two cholinergic pathways from the basal forebrain region to the hippocampus were identified in animals, one in the funbria-fornix system, and a second in the white matter of the temporal lobe. 22,24,29The anatomy of the human hippocampal cholinergic system is similar to that in rodents except for the absence of detectable intrinsic ChAT-immunoreactive perikarya. 16,ls'2s'37,39,a3,45The presence of gross, unbeaded ChAT-immunoreactive fibers in the fimbria and the parahippocampal gyrus and the absence of CHATimmunoreactive intrinsic perikarya suggest that the cholinergic innervation of the human hippocampus stems from extrinsic cholinergic perikarya, presumably located in the basal forebrain region. 27,33,39 *To whom correspondence should be addressed. Abbreviation: CHAT, choline acetyltransferase.
In Alzheimer's disease, both the cholinergic nuclei in the basal forebrain region27,~ and the cholinergic terminals in the hippocampal region ~1'~7'34'36'37'39-4' undergo degeneration. The hippocampi of patients with Alzheimer's disease also contain numerous senile plaques. Moreover, hippocampal pyramidal neurons are affected in Alzheimer's disease; high densities of neurofibrillary tangles and a loss of neurons are found in the hippocampal stratum pyramidale.5,6.19,3]'3s A previous study showed that the density of senile plaques in the hippocampus of patients with Alzheimer's disease is not related to the loss of cholinergic terminalsfl Whether or not the degeneration of hippocampal pyramidal neurons is related to the loss of cholinergic terminals is so far unclarified. We compared, therefore, the decrease of cholinergic terminals in the stratum pyramidale to the loss of pyramidal cells and the density of neurofibrillary tangles in the hippocampus of patients with Alzheimer's disease.
EXPERIMENTAL PROCEDURES
Characteristics of the patients and controls
The brains of four control subjects who had suffered from various non-neurological diseases and five patients with Alzheimer's disease exempt from vascular disease, brain tumor, infections and other neuropsychiatric disorders were studied (Table 1). Clinical diagnosis of Alzheimer's disease was based on the criteria of the DSM Ill. t The Alzheimer
843
G. RANSMAYR et al.
844
Table 1. Clinical and pathological characteristics of the patients
No. 1 2 3 4 5 6 7 8 9
Age (years)
Sex
Clinical diagnosis
76 75 78 92 67 70 87 80 83
f m f f m f f m f
control control control control Alzheimer Alzheimer Alzheimer Alzheimer Alzheimer
Disease duration (years) .... 3 3 15 5 1
Associated pathology Pulmonary embolism Bronchopneumonia Bladder cancer Pulmonary embolism Bronchopneumonia Bronchopneumonia Bronchopneumonia breast cancer
Post mortem delay (h)
Brain weight (g)
10 19 5 7 4 4 11 9 28
1210 1490 1220 940 980 1170 1150 1180
Temporal Frontal cortex cortex (SP/mm 2) (SP/mm 2) 1 0 1 0 72 64 40 52 54
3 I 0 0 66 60 16 49 43
For statistical evaluation see Experimental Procedures (Characteristics of the patients and controls). SP, senile plaques. patients had been totally dependent on others for the most basic activities of living. Ages and post mortem delays were not different in the Alzheimer and control groups (P = 0.807 and 0.806; Mann-Whitney U-test). The weights of the brains were significantly higher in the controls compared with the patients with Alzheimer's disease (P =0.029, Mann-Whitney U-test). Post mortem neuropathological examination using the Bodian silver impregnation technique demonstrated high densities o f neuritic plaques (Table 1) and neurofibrillary tangles (not shown) in the frontal and temporal neocortex (Brodmann areas 10 and 21) and the hippocampal formation, meeting the histological criteria for Alzheimer's disease. 2t Histopathological examination of the frontal and temporal cortex and the hippocampus of the controls revealed only sparse neuritic plaques (Table 1) and low densities of neurofibrillary tangles (not shown).
Tissue preparation and immunohistochemistrv of the hippocampus The brains were dissected, fixed, cryoprotected and deep-frozen, as described elsewhere. 39 The entire anteroposterior extents of the hippocampi were cut serially on a sliding microtome into 40-#m-thick frontal sections. ChAT immunohistochemistry was performed on sections taken every 2 mm with a polyclonal antiserum directed to human CHAT. 9 The antiserum to ChAT was well characterized and its specificity had been verified in previous studies. 4,7,9,23,39 The immunohistochemical procedures are described elsewhere) s'44 Sections adjacent to the ChAT-immunostained sections were stained with hematoxylin-eosin to exclude vascular lesions. Bodian's silver-impregnation technique was employed to visualize neuritic plaques and neurofibrillary tangles. Sections at a distance of 80/~m from those selected for ChAT immunohistochemistry were treated with Cresyl Violet to visualize neuronal perikarya. To demonstrate neurofibrillary tangles and amyloid plaques together with cholinergic structures, the ChAT-immunostained sections were counterstained with thioflavin S.42 Sections at a distance of 6 0 - 3 2 0 # m from the ChAT-immunostained sections were treated with a mouse monoclonal antibody against phosphorylated neurofilament (RT97). z3° The immunohistochemical procedures, including the dilution of the primary antibody, were identical to those described for ChAT immunohistochemistry except for the use of a goat anti-mouse secondary antibody (dilution 1:10) and a mouse peroxidase-antiperoxidase complex (dilution 1: 500). Histological structures quantified and regions analysed The entire anteroposterior extent of each hippocampus was divided into five segments of equal length. From each segment, a series of sections was selected to quantify in the stratum pyramidale ChAT-immunostained structures. RT97 and thioflavin S-stained neurofibrillary tangles, and Cresyl Violet-stained cell bodies.
Quantitative analyses of ChAT-immunoreactive structures, neurofibrillary tangles and neurons were performed in the CA4 and CA1 sectors and the subiculum. CA4, CAI and the subiculum were distinguished on Cresyl Violetstained sections according to criteria described elsewhere ~6.25 (Fig. 1). In brief, the extension of the stratum pyramidale into the area surrounded by the stratum granulare was defined as the CA4 sector. The transition from CA2 to CAI is characterized by a gradual widening of the stratum pyramidale. Moreover, in CAI the density o f pyramidal neurons is lower than in CA2, the cell bodies are larger ("giant neurons"), and the stratum pyramidale is divided into two main layers of neurons. The transition from the CA1 sector to the subiculum is characterized by a loss of the stratum radiatum of CA1, a gradual narrowing of the layer of hippocampal pyramidal cells and a widening of a subjacent layer of darker stained and less regularly shaped pyramidal neurons designated as subicular pyramids. 25 "'Clouds" of moderately stained and densely packed perikarya overlying the subicular pyramids mark the transition from the subiculum to the presubiculum.
Quantification of choline acetyltransferase-positive punctate immunoprecipitates The preponderant ChAT-immunostained structures in the stratum pyramidale were fine, beaded fibers and punctate immunoprecipitates characterized elsewhere as fiber varicosities and axon segments of cholinergic terminals t4:s'45 (Fig. 2A, C). The density of punctate ChAT-immunoprecipitates was taken as an index of the density of cholinergic terminals, t4:sm In order to verify whether ChAT-immunostained punctates were homogeneously distributed within the hippocampus and sampling methods could be used, one ChAT-immunostained section was selected from the central part of each hippocampus and analysed as follows. In CA4, CA1 and the subiculum, the longitudinal extent of the stratum pyramidale was divided into five parallel layers of equal width oriented in parallel with the outer circumference of the hippocampus. The five layers were spaced into 0.14 x 0.1 mm microscopic fields arranged consecutively at 0.14 mm intervals along the entire longitudinal extents of the three subregions. The fields were visualized at × 2000 magnification on a 24 × 20 cm videoscreen covered with two 18 x 18 mm square openings separated by 10cm (corresponding to two 9 x 9 # m microscopic fields separated by 50/~m). Quantification of ChAT-immunoreactive punctate structures was conducted on the 18 x 18 mm squares on the videoscreen. To analyse whether there were differences in the densities of ChAT-positive punctate immunoprecipitates between the five layers, one-way analysis of variance was performed on each of the nine selected sections separately in the three regions. No statistical differences were found between the five layers on any section in any region (F =2.116 to 0.19; P =0.117 to 0.941). Therefore. on the remaining sections of the nine brains examined,
Cholinergic system in Alzheimer hippocampi
845
Fig. 1. (A) Representative Cresyl Violet-stained frontal section (40/~m thick) through the mid-part of the anteroposterior extent of the hippocampus of control brain 4. The stratum pyramidale of CA4, CA1 and the subiculum is outlined according to the methods described in the text. PRE, presubiculum; SUB, subieulum. (B) Section at a distance of 80/am along the anteroposterior extent of the hippocampus from the section shown in A labeled with a polyclonal antibody specific for human placental CHAT.
the densities of ChAT-positive punctate immunoprecipitates were quantified only in the layer in the midline of the stratum pyranaidale with an otherwise identical method. Finally, the mean densities of ChAT-immunostained punctates were calculated for each subregion of each section.
Quantification of thioflavin S- and RT97-positive neurofibrillary tangles The thioflavin S-positive neurofibrillary tangles (Fig. 2E) were visualized under UV fight (Olympus, BH2-DMB filter block, green filter). Quantification of RT97 (Fig. 2F) and thioflavin S-stained tangles was performed on the five selected sections for each staining within the entire surface areas of CA4, CAI and the subiculum. The surface areas of the stratum pyramidale of the subregions were determined with a semiautomatic image analysing system (Histo 2000, Biocom, France). To obtain surface densities of neurofibrillary tangles for each subregion on each section, the number of tangles per subregion was divided by the surface areas.
Quantification of Cresyl Violet (Nissl)-stained neurons Staining with Cresyl Violet revealed, in the hippocampal pyramidal layers, triangular, ovoid or polygonal, blue perikarya (Fig. 2B, D). Quantification was performed with a x 40 objective (= x 400 magnification) in 0.25 x 0.25 mm fields spaced consecutively at 0.25 nun intervals along the entire longitudinal extents of the selected subregions in two layers centered in the inner and outer halves of the radial diameter of the stratum pyramidale. Only neurons containing a dark blue nucleolus were counted. Finally, the mean densities of neurons per mm 2 were calculated for each subregion of each section.
Statistical analysis The Friedman test was used to analyse the controls and the Alzheimer group separately, whether there were differences in the densities of ChAT-positive punctate immunoprecipitates, thiotlavin S- and RT97-positive tangles and Cresyl Violet-stained neurons in CA4, CA1 and the subiculum between the five levels of the anteroposterior extent of the hippocampus. No differences were found between the five levels in the two groups (Chi- squared scores ranging
from 1.2 to 8.7; P from 0.878 to 0.074). Therefore, the subregional density scores of the five sections of each hippocampus were averaged and the resultant mean densities of ChAT-positive punctate precipitates, neurofibrillary tangles and neurons for the Alzheimer and the control brains compared with the Mann-Whitney U-test. In addition, Kruskal-Wallis ANOVA and Mann-Whitney U-test were applied to compare, in the Alzheimer and the control groups separately, CA4, CA1 and the subiculum as to differences in the mean densities of the structures quantified with the three subregions. RESULTS
Choline acetyltransferase-positive punctate immunoprecipitates in the hippocampus of controls and patients with Alzheimer's disease In the controls, the densities of ChAT-positive punctate immunoprecipitates were significantly higher in CA4 than in CA1 and the subiculum, and in CA1 compared with the subiculum (Table 2). In the Alzheimer group, significantly higher densities of ChAT-positive punctate immunoprecipitates were found in CA4 and CA1 compared with the subiculum. The densities of ChAT-positive punctate immunoprecipitates in CA4, CA1 and the subiculum were significantly diminished in the Alzheimer group as compared to the controls (Table 2; Fig. 2A, C). There was a mean 32-33% loss of ChAT-positive terminals in the three subregions in the Alzheimer group. The decrease of ChAT-immunoreactive punctate structures in the Alzheimer group varied from patient to patient (almost no loss in patient 9 and marked loss in patient 6). In the individual patients, however, no major differences in the decrease of ChAT-positive punctate immunoprecipitate were found between the three subregions.
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Fig. 2. Photomicrographs taken from the CA1 sector, stratum pyramidale, of the mid-part of the anteroposterior extent of the hippocampus of control brain number 1 (A, B) and Alzheimer brain 7 (C-F). Scale bar = 50/zm. (A) ChAT immunohistochemistry revealing in the normal control brain networks of fine, beaded fibers and dense punctate immunoprecipitates characterized elsewhere as cholinergic terminals (see text). (B) Cresyl Violet-staining of a control brain section revealing hippocampal pyramidal neurons. The arrow points to a nucleolus. (C) ChAT immunohistochemistry in Alzheimer's disease showing remnants of punctate immunoprecipitates and fine and beaded fibers with partly globular enlargements. These changes of cholinergle terminals are found characteristically in Alzheimer's disease (arrows). (D) Cresyl Violet-stained pyramidal neurons in an Alzheimer brain. The arrow points to a nucleolus. (E) Dark-field photomicrograph (UV light, BH2-DMB filter block, Olympus) of the microscopic field shown in bright-field in C, counterstained with thioflavin S to demonstrate neurofibrillary tangles (long arrows). The short arrow points to the ChAT-immunoreactive beaded fiber visualized in C. (F) Neurofibrillary tangles visualized immunohistochemically with a monoclonal antibody to phosphorylated neurofilament (RT97).
Neurofibrillary tangles in the hippocampus of controls and patients with Alzheimer's disease In the controls, the densities of thioflavin S-stained neurofibrillary tangles (Fig. 2E) did not differ between CA4, CA1 and subiculum (Table 3). In the Alzheimer group, density of thioflavin S-stained neurofibrillary tangles was higher in CAI compared
to CA4, whereas no differences were found between CA1 and the subiculum, and CA4 and the subiculum. In the three regions analysed the densities of thioflavin S-stained tangles were significantly higher in the brains of patients with Alzheimer's disease than in controls (Table 3). The densities of thioflavin S-stained neurofibrillary tangles in CA4 and CA1 were higher in brains 6 and 7 than in 5, 8 and 9.
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Cholinerglc system in Alzheimer hippoeampi Table 2. Density of choline acetyltransferase-immunoreaetive punetate precipitates (eholinergic terminals) in the human hippocampal stratum pyramidale Brain number
Clinical diagnosis
CA4
CA 1
Subiculum
1 2 3 4 Mean
control control control control
52.7 __+5.7 45.5 + 5.5 47.3 _ 1.2 52.4 _ 6.7 49.5 + 1.8
41.5 -I- 4.2 40.7 _ 2.9 32,4 + 2.4 42,5 + 2.4 39.3 _ 2.4
28.9 -I- 4.3 25.8 __+3.0 22.9 _+ !.9 33.6 __+3.1 27.9 __+2.3
5 6 7 8 9 Meant
Alzheimer Alzheimer Alzheimer Alzheimer Alzheimer
26.2 __+1.9 20.7+2.1 28.6 + 3.3 39.1 __+1.6 50.8 _ 2.1 33.1 __+5.3*
27.6 __+2.8 19.2+ 1.7 24.6 + 1.6 31.8 __+1.8 29.1 _ 3.3 26.6 _ 2.5*
17.3 __+1.4 13.4+ 1.6 19.2 + 2.6 22.9_ 1.6 20.0 __+2.1 18.6 -+ 1.6"
Results are expressed as means + S.E.M. of 103 ChAT-immunoreactive punctate precipitates per mm 2 of five sections of the individual brains (see Experimental Procedures). Statistical analysis (only significant differences are indicated): interreglonal differences of the means of the controls and the Alzheimer group (Kruskal-Wallis ANOVA and Mann-Whitney U-test): controls, H = 9.27; CA4/CAI*, CA1/Sub*, CA4/Sub*; Alzheimer's disease, H = 7.31; CA1/Sub*, CA4/Sub*. tComparison of the means of CA4, CA1 and the subiculum of the Alzheimer group with those of the controls (Mann-Whitney U-test). *P
