Cholinergic innervation in neuritic plaques.

Although several studies of Alzheimer's disease suggest that the frequency of neuritic plaques in the cerebral cortex is correlated with the severity of dementia and with reduction in presynaptic cholinergic markers in the cortex, the relationship between cholinergic cortical innervation and the pathogenesis of plaques is unknown. The hypothesis was tested that the neurites in the plaque consist, in part, of presynaptic cholinergic axons, many of which arise from neurons in the basal forebrain. This hypothesis was tested by analyzing the character and distribution of plaques in monkeys, aged 4 to 31 years, with staining for acetylcholin-esterase and also with Congo red and silver stains. Immature and mature plaques were rich in acetylcholinesterase. As the plaques matured, the amount of amyloid increased, and the number of neurites and the activity of acetylcholinesterase decreased. End-stage amyloid-rich plaques lacked acetylcholinesterase. These observations indicate that changes in cortical cholinergic innervation are an important feature in the pathogenesis and evolution of the neuritic plaque.

Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain.

Recent evidence indicates that the nucleus basalis of Meynert, a distinct population of basal forebrain neurons, is a major source of cholinergic innervation of the cerebral cortex. Postmortem studies have previously demonstrated profound reduction in the presynaptic markers for cholinergic neurons in the cortex of patients with Alzheimer's disease and senile dementia of the Alzheimer's type. The results of this study show that neurons of the nucleus basalis of Meynert undergo a profound (greater than 75 percent) and selective degeneration in these patients and provide a pathological substrate of the cholinergic deficiency in their brains. Demonstration of selective degeneration of such neurons represents the first documentation of a loss of a transmitter-specific neuronal population in a major disorder of higher cortical function and, as such, points to a critical subcortical lesion in Alzheimer's patients.

Evidence for cholinergic neurites in senile plaques.

In the neocortices and amygdalae of young and aged macaques, cholinergic axons were identified by means of a monoclonal antibody to bovine choline acetyltransferase. Many fine, linear, immunoreactive profiles were seen in these animals. In the older animals, some cholinergic axons showed multifocal enlargements along their course. In some instances, neurites with choline acetyltransferase immunoreactivity were associated with deposits of amyloid (visualized with thioflavin T fluorescence). The appearance of these amyloid-associated abnormal cholinergic processes was similar to that of neurites in senile plaques, as shown by conventional silver impregnation techniques. Cholinergic systems thus give rise to some of the neurites within senile plaques.

Topography of the magnocellular basal forebrain system in human brain.

In primates, the large neurons in the nucleus basalis of Meynert (nbM), nucleus of the diagonal band of Broca (dbB), and medial septum are part of a cholinergic system with direct projections to amygdala, hippocampus, and cortex. Recent evidence indicates that neurons of this system selectively degenerate in individuals with Alzheimer's disease (AD) and suggests that degeneration of these cells contributes to the loss of presynaptic cortical cholinergic markers which occurs in these patients. The present report describes the topographical distribution of these large intensely basophilic, basal forebrain neurons in human brain. Rostrally, neurons of this magnocellular system are present in the medial septum and the dorsal and ventral parts of the nucleus of the dbB. The largest number occur in the nbM, which is situated in the substantia innominata below the globus pallidus. Caudally, large nbM-type neurons are found along the ventral and lateral edges of the globus pallidus. Neurons of this type are also encountered in the white matter below the putamen and nucleus accumbens, at the edges of the anterio commissure, in the white matter laminae of the globus pallidus and within and at the medial edge of the genu of the interal capsule. Directions for dissection of this system in human brain are given in an Appendix.

Multiple transmitter systems contribute neurites to individual senile plaques.

Senile plaques (SP), which consist largely of abnormal neuronal processes in proximity to deposits of amyloid, are a characteristic neuropathological feature of Alzheimer's disease. In lesser numbers, SP also occur in the brains of nondemented aged humans and nonhuman primates. To date, it is not known whether neurites in individual SP derive from neurons of one or several neurotransmitter systems. In aged monkeys, two strategies were used to test the hypothesis that individual SP can contain abnormal neurites arising from multiple neuronal systems. First, immunocytochemical methods were used to identify somatostatin-immunoreactive neurites in plaques, and these sections were subsequently stained with silver to visualize other neurites. Numerous plaques contained both somatostatin-positive and somatostatin-negative (i.e. argyrophilic only) neurites, suggesting that more than one transmitter system contributed neurites to each of these plaques. Second, two-color immunocytochemical techniques showed, in a small percentage of plaques, that cholinergic neurites coexist with neuropeptide Y (NPY)-containing neurites or catecholaminergic neurites. These results suggest that the formation of SP may result from events that involve abnormalities of neuronal processes arising from multiple transmitter systems.

Phosphorylated neurofilament antigens in neurofibrillary tangles in Alzheimer's disease.

Neurofibrillary tangles (NFT) are a hallmark of Alzheimer's disease (AD), and their presence correlates with the presence of dementia. A major constituent of NFT is the insoluble paired helical filament which shares some antigenic relationships with normal cytoskeletal elements, particularly neurofilaments. If neurofilament proteins (200, 145-160, and 68 kilodaltons [kd]) participate in the formation of NFT, the distribution of these constituents might be expected to be abnormal. To examine this issue, we used immunocytochemical methods to localize phosphorylated and nonphosphorylated epitopes of neurofilament proteins in hippocampal neurons of controls and patients with AD. Normally, the 200-kd neurofilament protein is not phosphorylated in the perikarya of neurons. However, in AD, many pyramidal neurons contained immunoreactive phosphorylated neurofilaments. Patterns of immunoreactivity (linear, flame-shaped, or skein-like within perikarya) greatly resembled the appearance of silver-stained NFT. This pattern of immunoreactivity was not present in hippocampal pyramidal neurons in controls, except in one aged patient in whom adjacent silver-stained sections revealed a few NFT. Patterns of immunoreactivity with antibodies for nonphosphorylated neurofilament proteins were similar in control and AD neurons. Our results indicate that some NFT are associated with abnormal distributions of high molecular weight phosphorylated neurofilament proteins. One domain of the 200-kd protein is believed to be a component of the side arms which link neurofilaments and interact with microtubules. Abnormal interactions of perikaryal neurofilaments could play a role in the genesis of NFT, and this abnormality of the cytoskeleton could contribute to the dysfunction of neurons at risk in AD.

Neuropeptidergic systems in plaques of Alzheimer's disease.

Polyclonal antibodies directed against substance P, somatostatin, neurotensin, cholecystokinin (CCK), leucine enkephalin, and vasoactive intestinal polypeptide (VIP) were employed to determine the immunoreactivities of neurites of senile plaques (SP) in Alzheimer's disease (AD). All of the antibodies labeled some neurites in some SP. The transmitter specificities of immunoreactive neurites tended to reflect the distribution of transmitter-associated fibers in normal tissue. This investigation also documented the presence of abnormal axons (as distinct from neurites within plaques) in the neuropil in brains of individuals with AD and in some aged controls. These findings suggest that a variety of transmitter systems are involved in the formation of neuropil abnormalities of SP. They also indicate that a greater number of neuronal systems are affected in AD than have been documented by neurochemical studies.

Hippocampal lesions in dominantly inherited Alzheimer's disease.

We compared hippocampal lesions in three pedigrees of Familial Alzheimer's Disease (FAD). In these pedigrees, the disease is inherited as an autosomal dominant disorder and has been linked to DNA markers on chromosome 21. In eight cases of FAD (four from one pedigree and two each from two others) we quantified neurofibrillary tangles (NFT) and senile plaques (SP) in hippocampal subdivision CA1-4, subiculum, presubiculum, and dentate gyrus. We observed consistent patterns of the distribution of lesions: The highest density of NFT and SP was present in CA1-2; virtually no SP or NFT were present in presubiculum; SP diameter was consistently greatest in CA4. We found no overall differences among pedigrees in total densities of NFT and SP, but statistical analyses disclosed that an uncommon type of SP was disproportionately present in two pedigrees. This type of SP was usually restricted to CA4, had a marked amyloid core devoid of argyrophilic neurites. These studies also disclosed inter- and intrafamilial heterogeneity of lesion distribution (including congophilic angiopathy and cerebellar plaques) in these three pedigrees.

Olfactory bulb lesions in Alzheimer's disease.

The olfactory bulb (OB), with its comparatively simple and well-delineated connectivity, presents an interesting system for examining cell-specific pathology in neurologic degenerative disorders such as Alzheimer's disease (AD). We have found that in AD the large, efferently projecting neurons (mitral cells) of the OB degenerate, typically without classical Alzheimer neurofibrillary changes. In some cases, with less severe neocortical pathology, the terminal arborizations of olfactory nerve appear hyperplastic and are associated with focal accumulations of A-4 (beta-amyloid) immunoreactivity that are not detectable by standard amyloid stains. These abnormalities may represent a pathologic manifestation of normally occurring plasticity in the olfactory system.

Nicotinic cholinoceptive neurons of the frontal cortex are reduced in Alzheimer's disease.

The cellular distribution of nicotinic acetylcholine receptors was studied in the frontal cortex (area 10) of 1) Alzheimer patients and compared to 2) age-matched and 3) middle-aged controls using the monoclonal antibody WF 6 and an immunoperoxidase protocol. Statistical analysis revealed significant differences between the number of labeled neurons among all three groups tested (middle-aged controls greater than aged controls greater than Alzheimer cases). No differences were seen for cresyl violet-stained samples. These findings underline that the nicotinic receptor decrease found with radioligand binding may reflect a postsynaptic in addition to a presynaptic component.

Age-related changes in multiple neurotransmitter systems in the monkey brain.

Age-associated changes in cholinergic, monoaminergic and amino acid neurotransmitter systems were analyzed in 14 brain regions of 23 rhesus monkeys that ranged in age from 2 to 37 years. In the frontal pole, the levels of choline acetyltransferase (ChAT) activity, the density of [3H]ketanserin (serotonin type-2) binding sites and endogenous levels of dopamine, homovanillic acid and serotonin, all expressed per milligram of protein, decreased significantly with aging. In precentral motor cortex, ChAT activity decreased; in parietal and occipital cortex, the number of [3H]ketanserin binding sites decreased while the number of Na+-independent [3H]glutamate binding sites increased with age. In the caudate nucleus, endogenous levels of norepinephrine decreased. This descriptive study indicates that the aging monkey may be a very useful model for future investigations of age-associated transmitter abnormalities similar to those that occur in humans.

Acetylcholinesterase activity in senile plaques of aged macaques.

A modified acetylcholinesterase (AChE)-histochemical technique, which demonstrates axonal morphology to a high degree, was used to examine the neocortices of aged monkeys. This approach disclosed slender linear axonal profiles in young animals. In older monkeys, there was a variety of abnormalities of AChE-containing fibers, including multifocal distensions of individual fibers and aggregations of neurite-sized, AChE-rich swellings. Combined with thioflavin-T staining to visualize amyloid, this histochemical technique showed that some of these AChE-containing fibers were present in proximity to deposits of amyloid. This association suggests that abnormal AChE-rich axons participate in the formation of some senile plaques in the neocortices of aged nonhuman primates. While it is probable that many of these AChE-rich fibers are axons of cholinergic neurons residing in the basal forebrain, it is also likely that some of these fibers are derived from noncholinergic neuronal populations known to synthesize AChE. Immunocytochemical strategies can be used to assess the involvement of other systems, including cholinergic, noradrenergic, dopaminergic, somatostatinergic, and serotonergic neurons in the formation of senile plaques in the brains of aged nonhuman primates.

The neural basis of memory decline in aged monkeys.

Nonhuman primates experience changes in behavior as they progress into old age. Visual recognition, spatial learning, habit formation, and visuospatial manipulation are impaired in aged rhesus monkeys relative to young controls. We have begun to study the possible neural substrate for these changes, focusing on brain areas that are known, from lesion studies, to be essential for the successful performance of specific tasks. Aged nonhuman primates develop senile plaques, most commonly in amygdala, hippocampus, and neocortex. Our preliminary data suggest that the density of plaques may be related to poor behavioral performance in some aged monkeys. However, behavioral decline begins before the appearance of significant numbers of senile plaques, suggesting that other factors may interfere with cognition. Numerous studies of several genera have shown that receptors for neurotransmitters decline in number between the adolescent years and old age. Our autoradiographic analyses of primate temporal neocortex demonstrate loss of muscarinic, nicotinic, dopaminergic and serotoninergic receptor binding sites between the ages of 2 and 22 years. Preliminary data indicate that markers for adenyl cyclase and phosphatidyl inositol second-messenger systems also are reduced in temporal cortex. Although these declines represent a potential substrate for behavioral changes, no studies have directly related a decrease in receptor number to deficits in learning and memory in aged primates. Other changes in the aging brain include loss of neurons, reduced neurochemical markers, and decreased content of neuronal ribonucleic acid (RNA). All of these decrements may be interrelated to some extent in that decreased RNA could result in changes in neurochemical markers and receptors and, eventually, in dysfunction and death of neurons. These observations underscore the importance of establishing a time course for age-associated neural abnormalities, examining regions of brain in which changes are most likely to occur, and studying their relationship to the progression of behavioral dysfunction. Detailed anatomical analyses of the distribution of in situ uptake/receptor binding sites and messenger RNA (mRNA) in aged nonhuman primates may clarify some of the factors that most likely contribute to behavioral changes in elderly humans.

Age differences in recognition memory of the rhesus monkey (Macaca mulatta).

Aging is accompanied by a gradual decline in memory in both humans and nonhuman primates. To determine whether the impairment in nonhuman primates extends to recognition memory, which is a sensitive index of the integrity of the limbic system, we trained rhesus monkeys of four different age groups (3-6, 14-17, 20-24, and 25-29 years of age) on a delayed nonmatching-to-sample task with trial-unique objects. After the animals had learned the task, which required recognition of single objects presented ten seconds earlier, memory demands were increased by gradually lengthening delay intervals (to 120 seconds) and list lengths (to ten objects). With increasing age, only marginal impairments in learning the basic task were observed. However, clear age-related differences did emerge when either delays or list lengths were increased, with the oldest group of monkeys demonstrating the greatest impairments. The decline in visual recognition ability in aging monkeys parallels the decline in memory observed with advancing age in humans.

Hemianopsia in dementia with Lewy bodies.

OBJECTIVE: To describe the pathologic changes that caused a left homonymous hemianopsia in a patient with dementia with Lewy bodies. DESIGN: Report of a case and postmortem studies. MAIN OUTCOME AND RESULTS: A 66-year-old woman experienced parkinsonism and left homonymous hemianopsia early in the course of a rapidly progressive dementia that culminated in death only 21 months after the onset of her symptoms. Postmortem examination revealed pathologic features consistent with the diagnosis of dementia with Lewy bodies. The only apparent explanation for her visual field deficit was a disproportionately large number of neurofibrillary tangles in the right striate, peristriate, and inferotemporal cortices. CONCLUSION: A clinically obvious homonymous hemianopsia can result from the occipital and inferotemporal cortical degeneration in dementia with Lewy bodies.

Normal pattern of labeling of cerebral cortical corticotropin-releasing factor (CRF) receptors in Alzheimer's disease: evidence from chemical cross-linking studies.

Recent data have demonstrated that in Alzheimer's disease, the concentrations of corticotropin-releasing factor (CRF) were reduced and that there were reciprocal increases in CRF receptors in affected cerebrocortical areas. In order to determine whether the increases in CRF receptors in Alzheimer's disease were due to altered molecular composition of the binding protein, we compared the labeling pattern of 125I-Tyr0-ovine CRF in temporal neocortex of Alzheimer's patients and age-matched controls using chemical cross-linking techniques. A similar pattern of 125I-Tyr0-ovine CRF labeling was seen in Alzheimer's and control brains, with the major CRF binding protein corresponding to an apparent molecular weight of 58,000 Da. These data indicate that the increased CRF receptor population in cerebral cortex in Alzheimer's disease comprises bona fide CRF receptor binding subunits with no apparent change in the molecular structure.

Innervation of human hippocampus by noradrenergic systems: normal anatomy and structural abnormalities in aging and in Alzheimer's disease.

Immunocytochemical studies, using an antibody directed against human dopamine beta-hydroxylase, identified an extensive plexus of noradrenergic axons/terminals in normal human hippocampus. In hippocampi of individuals with Alzheimer's disease, the density of noradrenergic innervation was reduced and abnormal noradrenergic axons, which exhibited multifocal enlargements, were present in the neuropil. Some of these neurites were clustered around deposits of amyloid (senile plaques), and these abnormalities were most common in CA3-4, a region normally showing a relatively high density of noradrenergic terminals. This investigation provides direct evidence for structural abnormalities of noradrenergic axons/nerve terminals in hippocampi of individuals with Alzheimer's disease.

Catecholaminergic neurites in senile plaques in prefrontal cortex of aged nonhuman primates.

Immunocytochemical studies, using a polyclonal antibody directed against tyrosine hydroxylase, identified catecholaminergic axons in prefrontal cortex of young and aged nonhuman primates. Aged monkeys, who showed cortical senile plaques in silver stains, had swollen tyrosine hydroxylase-immunoreactive axons in neocortex. Some of these abnormal processes were associated with deposits of amyloid (visualized by thioflavin-T fluorescence) and were similar in appearance to neurites demonstrated by silver impregnation methods. This study provides evidence for structural abnormalities in catecholaminergic axons/nerve terminals in the neocortices of aged primates.

Effects of scopolamine and physostigmine on recognition memory in monkeys with ibotenic-acid lesions of the nucleus basalis of Meynert.

Monkeys with bilateral ibotenic-acid lesions of the nucleus basalis of Meynert, an area rich in cholinergic neurons that innervate the cerebral cortex, were compared with unoperated control monkeys on a recognition memory task. Although animals with large lesions had substantial reductions of cortical choline acetyltransferase activity, none showed impairment in the task. Lesion effects were observed, however, when performance was assessed following administration of a muscarinic receptor blocker (scopolamine) or a cholinesterase inhibitor (physostigmine). Although scopolamine produced dose-related impairments in both groups, this effect was greater in the experimental animals. Conversely, whereas physostigmine produced modest improvement in performance in the control group, no such improvement was observed in the experimental animals. The altered sensitivity to the mnemonic effects of cholinergic agents in the experimental group suggests that the cholinergic neurons of the nucleus basalis of Meynert contribute to recognition memory.

Cellular distribution and expression of cortical acetylcholine receptors in aging and Alzheimer's disease.

Ligand binding studies show marked reductions of nicotinic, but not of muscarinic binding sites in Alzheimer's disease. Using monoclonal antibodies we studied immunohistochemically the expression of the respective receptor proteins in the frontal cortex of middle-aged (55 +/- 5 yr) controls, age-matched controls (73 +/- 6 yr), and patients with Alzheimer's disease (74 +/- 5 yr). Density of nicotinic cholinoceptive neurons was 8000/mm3 for middle-aged controls and 4000/mm3 for age-matched controls, but only 900/mm3 in Alzheimer's brains (p less than 0.0001). Densities of muscarinic cholinoceptive and of Nissl-stained neurons were not significantly different between the groups, pointing to a selective decrease of nicotinic receptor protein expression in cortical neurons with aging and in Alzheimer's disease.