Cholinergic neuronal and axonal abnormalities are present early in aging and in Alzheimer disease

A large body of evidence indicates that basal forebrain cholinergic neurons are selectively vulnerable to degeneration early in Alzheimer disease (AD). Recent studies, however, demonstrate reductions in cortical activity of the cholinergic enzyme choline acetyltransferase only in late stages of AD. To address this apparent contradiction, we compared abnormalities in magnocellular basal forebrain cholinergic neurons and their axons in nondemented young (<65 years; n = 6), nondemented old (>65 years; n = 7), pathologically mild (n = 5), and pathologically severe (n = 5) AD cases. Cholinergic axon abnormalities (i.e. thickened fibers and ballooned terminals) were evident in nondemented middle-aged cases, increased in nondemented old cases, and reduced in density in severe AD. This suggests that loss of cortical cholinergic axons in AD occurs preferentially in fibers with these abnormalities. Paired helical filament 1-immunoreactive pretangles and tangles were observed as early as the third decade prior to their appearance in entorhinal/perirhinal cortex; they were increased in mild and severe AD. These results indicate that basal forebrain cholinergic neuron abnormalities are present very early in aging and in the course of AD. Therefore, despite the morphologic alterations, choline acetyltransferase activity, but not necessarily normal neuron functions, may be preserved.     

Depletion of cholinergic neurons in the nucleus of the medial septum and the vertical limb of the diagonal band in dementia with Lewy bodies

The cholinergic basal forebrain is divided into four subregions (Ch1–4), and cholinergic neuronal loss in the nucleus basalis of Meynert (Ch4) has been correlated with cognitive impairments in both Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB). However, the Ch1–2 regions, which provide the major cholinergic innervation to the hippocampus, have not been investigated in DLB. The purpose of this study was to reveal the cholinergic neuronal changes in the medial septum (Ch1) and the nucleus of the vertical limb of the diagonal band (Ch2) of DLB brains. Using choline acetyltransferase (ChAT) immunohistochemistry, we showed that the number of ChAT-immunoreactive neurons in DLB brains was significantly lower than the numbers in AD and non-demented (control) brains. No significant difference in the number of ChAT-immunoreactive neurons was found between the AD and control brains. Moreover, the size of the ChAT-immunoreactive neurons was significantly smaller in the AD and DLB brains than in the control brains. These results show that cholinergic neurons of the Ch1-2 regions are more severely affected in DLB than in AD. Our DLB cases did not fulfill the neuropathologic criteria for definite AD. Furthermore, some Lewy bodies were observed in the Ch1-2 regions. Thus, cholinergic neuronal loss in the Ch1-2 regions might be specific to the pathology of DLB. Taking the distribution of cholinergic fibers in the hippocampus into consideration, this study suggests a possibility that hippocampal cholinergic projection is involved in Lewy-related neurites in the CA2–3 regions, the origin of which remains unclear.     

Regional distribution of choline acetyltransferase and acetylcholinesterase within the amygdaloid complex and stria terminalis system

The distribution of ‘marker’ enzymes for cholinergic neurons has been studied in 10 subdivisions of the amygdaloid complex of the rat brain. Choline acetyltransferase activity was measured using a radiochemical method in samples dissected from fresh serial sections. Acetylcholinesterase was studied using a histochemical procedure. Both enzymes had similar patterns of distribution within the amygdaloid complex and were most concentrated in the posterior lateral and basolateral nuclei and in the nucleus of the lateral olfactory tract. These enzymes were much less concentrated in the cortical, medial, central, and basomedial nuclei. Large differences in acetylcholinesterase staining were found within the lateral posterior and the basolateral nuclei and within the pyriform cortex. Biochemical studies showed a parallel distribution of choline acetyltransferase within these nuclei. The results indicate that cholinergic neural elements in the amygdala are concentrated primarily in the basolateral complex and suggest that this region may be innervated by cholinergic fibers traveling in the ventral amygdalo-fugal pathway.     

Atrophy of the cholinergic basal forebrain in dementia with Lewy bodies and Alzheimer’s disease dementia

Similar to Alzheimer’s disease (AD), dementia with Lewy bodies (DLB) is characterized by a profound degeneration of cortically-projecting cholinergic neurons of the basal forebrain (BF) and associated depletion of cortical cholinergic activity. We aimed to investigate subregional atrophy of the BF in DLB in vivo and compare it to the pattern of BF atrophy in AD. Structural MRI scans of 11 patients with DLB, 11 patients with Alzheimer’s disease, and 22 healthy controls were analysed using a recently developed technique for automated BF morphometry based on high-dimensional image warping and cytoarchitectonic maps of BF cholinergic nuclei. For comparison, hippocampus volume was assessed within the same morphometric framework using recently published consensus criteria for the definition of hippocampus outlines on MRI. The DLB group demonstrated pronounced and subregion-specific atrophy of the BF which was comparable to BF atrophy in AD: volume of the nucleus basalis Meynert was significantly reduced by 20–25 %, whereas rostral BF nuclei were only marginally affected. By contrast, hippocampus volume was markedly less affected in DLB compared to AD. Global cognition as determined by MMSE score was associated with BF volume in AD, but not in DLB, whereas visuoperceptual function as determined by the trail making test was associated with BF volume in DLB, but not in AD. DLB may be characterized by a more selective degeneration of the cholinergic BF compared to AD, which may be related to the differential cognitive profiles in both conditions.     

Estradiol increases choline acetyltransferase activity in specific basal forebrain nuclei and projection areas of female rats

Administration of estradiol to gonadectomized female, but not male rats, is associated with increased activity of choline acetyltransferase in the medial aspect of the horizontal diagonal band nucleus, the frontal cortex, and CA1 of the dorsal hippocampus. Four other basal forebrain cholinergic nuclei did not show changes in choline acetyltransferase activity after estradiol. These data have implications for possible benefits of estradiol administration to patients with senile dementia of the Alzheimer's type.     

Loss of medial septum cholinergic neurons in THY-Tau22 mouse model: what links with tau pathology?

Alzheimer's disease (AD) is a neurodegenerative disorder histologically defined by the cerebral accumulation of amyloid deposits and neurofibrillary tangles composed of hyperphosphorylated tau proteins. Loss of basal forebrain cholinergic neurons is another hallmark of the disease thought to contribute to the cognitive dysfunctions. To this date, the mechanisms underlying cholinergic neurons degeneration remain uncertain. The present study aimed to investigate the relationship between neurofibrillary degeneration and cholinergic defects in AD using THY-Tau22 transgenic mouse model exhibiting a major hippocampal AD-like tau pathology and hyperphosphorylated tau species in the septohippocampal pathway. Here, we report that at a time THY-Tau22 mice display strong reference memory alterations, the retrograde transport of fluorogold through the septohippocampal pathway is altered. This impairment is associated with a significant reduction in the number of choline acetyltransferase (ChAT)-immunopositive cholinergic neurons in the medial septum. Analysis of nerve growth factor (NGF) levels supports an accumulation of the mature neurotrophin in the hippocampus of THY-Tau22 mice, consistent with a decrease of its uptake or retrograde transport by cholinergic terminals. Finally, our data strongly support that tau pathology could be instrumental in the cholinergic neuronal loss observed in AD.     

In situ hybridization localization of choline acetyltransferase mRNA in adult rat brain and spinal cord

The cellular distribution of choline acetyltransferase (ChAT) mRNA within the adult rat central nervous system was evaluated using in situ hybridization. In forebrain, hybridization of a ³⁵S-labeled rat ChAT cRNA densely labeled neurons in the well-characterized basal forebrain cholinergic system including the medial septal nucleus, diagonal bands of Broca, nucleus basalis of Meynert and substantia innominata, as well as in the striatum, ventral pallidum, and olfactory tubercle. A small number of lightly labeled neurons were distributed throughout neocortex, primarily in superficial layers. No cellular labeling was detected in hippocampus. In the diencephalon, dense hybridization labeled neurons in the ventral aspect of the medial habenular nucleus whereas cells in the lateral hypothalamic area and supramammillary region were more lightly labeled. Hybridization was most dense in neurons of the motor and autonomic cranial nerve nuclei including the oculomotor, Edinger-Westphal, and trochlear nuclei of the midbrain, the abducens, superior salivatory, trigeminal, facial and accessory facial nuclei of the pons, and the hypoglossal, vagus, and solitary nuclei and nucleus ambiguus of the medulla. In addition, numerous cells in the pedunculopontine and laterodorsal tegmental nuclei, the ventral nucleus of the lateral lemniscus, the medial and lateral divisions of the parabrachial nucleus, and the medial and lateral superior olive were labeled. Occasional labeled neurons were distributed in the giantocellular, intermediate, and parvocellular reticular nuclei, and the raphe magnus nucleus. In the medulla, light to moderately densely labeled cells were scattered in the nucleus of Probst's bundle, the medial vestibular nucleus, the lateral reticular nucleus, and the raphe obscurus nucleus. In spinal cord, the cRNA densely labeled motor neurons of the ventral horn, and cells in the intermediolateral column, surrounding the central canal, and in the spinal accessory nucleus. These results are in good agreement with reports of the immunohistochemical localization of ChAT and provide further evidence that cholinergic neurons are present within neocortex but not hippocampus.     

Choline acetyltransferase activity in the cerebellum and in centers of lateral line system of teleosts

Using choline acetyltransferase as a marker of cholinergic activity, different cerebellar areas and brainstem nuclei were assayed in the goldfish and the catfish. Enzyme activity resulted remarkedly higher in archicerebellum than in paleocerebellum. In addition three brainstem nuclei related to lateral line system, showed high or very high levels of choline acetyltransferase. The results suggest that cholinergic transmission would play an important role in central circuits of lateral line system, including the projection towards archicerebellar areas.     

Long-term effects of basal forebrain lesions on cholinergic, noradrenergic and serotonergic markers in mouse neocortex

Lesions of basal forebrain cholinergic neurons projecting to cerebral cortex and hippocampus have recently been exploited as animal models for some of the neurochemical and behavioral deficits of Alzheimer's disease. We have observed that electrolytic lesions of cholinergic basal forebrain nuclei can lead to morphological plasticity in adult mouse cortex. In the present study, the acute and chronic sequelae of basal forebrain electrolytic lesion on cortical synaptic chemistry have been examined. In addition to choline acetyltransferase (ChAT) activity, levels of norepinephrine and of serotonin were reduced within a week after the lesion. Recovery of ChAT activity and of serotonin levels began within a month after the lesion. Serotonin type 2 receptor binding exhibited an acute reduction after the lesion in ipsilateral cortex, followed later by a chronic bilateral decrease. No significant changes in beta-adrenergic receptors were apparent at any time after the lesion despite a permanent and bilateral reduction of norepinephrine levels after the lesion. The potential significance of these results for cortical plasticity regulation and Alzheimer's disease is discussed.     

Cholinergic and catecholaminergic afferents to the olfactory bulb in the hamster: a neuroanatomical, biochemical, and histochemical investigation

A series of neuroanatomical, biochemical, and histochemical studies have been conducted to determine the sources of cholinergic afferents to the main olfactory bulb (MOB) in the hamster. Following horseradish peroxidase (HRP) injections that are restricted to the MOB, retrograde neuronal labeling is observed bilaterally in the anterior olfactory nucleus, locus coeruleus, and raphe nuclei, and ipsilaterally in the ventral hippocampal rudiment, dorsal peduncular cortex, piriform cortex, nucleus of the lateral olfactory tract, anterior pole of the medial septal area and vertical limb of the diagonal band, nucleus of the horizontal limb of the diagonal band (HDB), and hypothalamus. Spread of HRP into the accessory olfactory bulb results in additional neuronal labeling ipsilaterally in the bed nucleus of the accessory olfactory tract, medial amygdaloid nucleus, and bed nucleus of the stria terminalis, and bilaterally in the posteromedial cortical amygdaloid nucleus. Retrograde tracing studies also have been conducted in cases with lesions in the basal forebrain or hypothalamus to assess the extent to which such lesions interrupt fibers of passage from other sources of centrifugal afferents, and the effects of such lesions on choline acetyltransferase (CAT) activity and catecholamine content in the MOB and on acetylcholinesterase (AChE) activity in the forebrain have been evaluated. Lesions in the basal forebrain reduce or eliminate CAT and AChE activity in the MOB in direct relationship to the extent of damage to the HDB. Norepinephrine (NE) content in the MOB also is reduced by basal forebrain lesions, but in relationship to damage of the medial forebrain bundle (MFB). The hypothalamic lesions have no effect on AChE activity in the forebrain or on CAT activity in the MOB, but they eliminate retrograde labeling in the locus coeruleus and raphe nuclei and reduce the NE content of the MOB to undetectable levels. The dopamine content of the MOB is not reduced by any of the lesions. Anterograde tracing studies have been conducted to compare the rostral projection patterns of the HDB with the distribution of AChE activity. Most of the rostrally directed axons travel in association with the MFB. A small component of axons travels in association with the lateral olfactory tract. Within the MOB, the axons terminate predominantly in the glomerular layer and in the vicinity of the internal plexiform layer. The projection and termination patterns of the HDB correspond well with the distribution of AChE activity. These various results indicate that the HDB is the major source of cholinergic afferents to the MOB.     

Cholinergic innervation of the human thalamus: dual origin and differential nuclear distribution.

The cholinergic innervation of the human thalamus was studied with antibodies against the enzyme choline acetyltransferase (ChAT) and nerve growth factor receptor (NGFr). Acetylcholinesterase histochemistry was used to delineate nuclear boundaries. All thalamic nuclei displayed ChAT-positive axons and varicosities. Only the medial habenula contained ChAT-positive perikarya. Some intralaminar nuclei (central medial, central lateral, and paracentral), the reticular nucleus, midline nuclei (paraventricular and reuniens), some nuclei associated with the limbic system (anterodorsal nucleus and medially situated patches in the mediodorsal nucleus) and the lateral geniculate nucleus displayed the highest density of ChAT-positive axonal varicosities. The remaining sensory relay nuclei and the nuclei interconnected with the motor and association cortex displayed a lower level of innervation. Immunoreactivity for NGFr was observed in cholinergic neurons of the basal forebrain but not in cholinergic neurons of the upper brainstem. The contribution of basal forebrain afferents to the cholinergic innervation of the human thalamus was therefore studied with the aid of NGFr-immunoreactive axonal staining. The anterior intralaminar nuclei, the reticular nucleus, and medially situated patches in the mediodorsal nucleus displayed a substantial number of NGFr-positive varicose axons, presumably originating in the basal forebrain. Rare NGFr-positive axonal profiles were also seen in many of the other thalamic nuclei. These observations suggest that thalamic nuclei affiliated with limbic structures and with the ascending reticular activating system are likely to be under particularly intense cholinergic influence. While the vast majority of thalamic cholinergic input seems to come from the upper brainstem, the intralaminar and reticular nuclei, and especially medially situated patches within the mediodorsal nucleus also appear to receive substantial cholinergic innervation from the basal forebrain.     

Direct catecholaminergic-cholinergic interactions in the basal forebrain. II. Substantia nigra-ventral tegmental area projections to cholinergic neurons

Previous observations indicate that the basal forebrain receives dopaminergic input from the ventral midbrain. The present study aimed at determining the topographic organization of these projections in the rat, and whether this input directly terminates on cholinergic neurons. Injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) into discrete parts of the ventral tegmental area (VTA) and the substantia nigra pars compacta (SNC) labeled axons and terminals in distinct parts of the basal forebrain, including medial and lateral septum, diagonal band nuclei, ventral pallidum, globus pallidus, substantia innominata, globus pallidus, and internal capsule, where PHA-L-labeled terminals abutted cholinergic (choline acetyltransferase=ChAT-containing) profiles. Three—dimensional (3-D) computerized reconstruction of immunostained sections clearly revealed distinct, albeit overlapping, subpopulations of ChAT-immunoreactive neurons apposed by PHA-L-labeled input from medial VTA (mainly in vertical and horizontal diagonal band nuclei), lateral VTA and medial SNC (ventral pallidum and anterior half of substantia innominata), and lateral SNC (caudal half of the substantia innominata and globus pallidus). At the ultrastructural level, about 40% of the selected PHA-L-labeled presynaptic terminals in the ventral pallidum and substantia innominata were found to establish synaptic specializations with ChAT-containing profiles, most of which on the cell body and proximal dendritic shafts. Convergent synaptic input of unlabeled terminals that formed asymmetric synapses with the ChAT-immunoreactive profiles were often found in close proximity to the PHA-L-labeled terminals. These observations show that the cholinergic neurons in the basal forebrain are targets of presumably dopaminergic SNC/VTA neurons, and suggest a direct modulatory role of dopamine in acetylcholine release in the cerebral cortical mantle. © 1996 Wiley-Liss, Inc.     

Retrograde changes in the nucleus basalis of the rat, caused by cortical damage, are prevented by exogenous ganglioside GM1

In rats with extensive unilateral cortical damage, retrograde effects upon the cholinergic cells of the basal nucleus were observed. Cells of the basal nucleus stained immunocytochemically for choline acetyltransferase were shrunken and choline acetyltransferase enzymatic activity in that region was reduced. Both these effects could be prevented by the administration of the ganglioside GM1.     

The cholinergic system in mild cognitive impairment and Alzheimer's disease: An in vivo MRI and DTI study

Few studies have investigated in vivo changes of the cholinergic basal forebrain in Alzheimer's disease (AD) and amnestic mild cognitive impairment (MCI), an at risk stage of AD. Even less is known about alterations of cortical projecting fiber tracts associated with basal forebrain atrophy. In this study, we determined regional atrophy within the basal forebrain in 21 patients with AD and 16 subjects with MCI compared to 20 healthy elderly subjects using deformation‐based morphometry of MRI scans. We assessed effects of basal forebrain atrophy on fiber tracts derived from high‐resolution diffusion tensor imaging (DTI) using tract‐based spatial statistics. We localized significant effects relative to a map of cholinergic nuclei in MRI standard space as determined from a postmortem brain. Patients with AD and MCI subjects showed reduced volumes in basal forebrain areas corresponding to anterior medial and lateral, intermediate and posterior nuclei of the Nucleus basalis of Meynert (NbM) as well as in the diagonal band of Broca nuclei (P < 0.01). Effects in MCI subjects were spatially more restricted than in AD, but occurred at similar locations. The volume of the right antero‐lateral NbM nucleus was correlated with intracortical projecting fiber tract integrity such as the corpus callosum, cingulate, and the superior longitudinal, inferior longitudinal, inferior fronto‐occipital, and uncinate fasciculus (P < 0.05, corrected for multiple comparisons). Our findings suggest that a multimodal MRI‐DTI approach is supportive to determine atrophy of cholinergic nuclei and its effect on intracortical projecting fiber tracts in AD. Hum Brain Mapp, 2010. © 2010 Wiley‐Liss, Inc.     

Cholinergic innervation of the monkey amygdala: an immunohistochemical analysis with antisera to choline acetyltransferase

The organization of the cholinergic innervation of the macaque monkey amygdaloid complex was investigated by means of immunohistochemical techniques and either a polyclonal antiserum or a monoclonal antibody directed against the specific synthetic enzyme choline acetyltransferase (ChAT). Adjacent series of sections were processed histochemically for the demonstration of the degradative enzyme acetylcholinesterase (AChE) or for cell bodies with thionin. The density of ChAT immunoreactivity differed substantially among the various nuclei and cortical regions of the amygdala. In general, the distribution of ChAT immunoreactivity paralleled the pattern of AChE staining. One notable exception was the presence of AChE containing cell bodies in addition to AChE positive fibers within nearly all of the nuclear and cortical regions. In contrast, ChAT immunoreactivity was associated only with fibers and terminals. The highest density of ChAT immunoreactive fibers and terminals was consistently observed in the magnocellular subdivision of the basal nucleus. Staining was substantially less dense in the more ventrally situated parvicellular subdivision. Medially, in the adjacent accessory basal nucleus, immunoreactive fibers and terminals were densest in the magnocellular and superficial subdivisions and least prominent in the parvicellular subdivision. Of the deep nuclei, the lateral nucleus generally obtained the least ChAT immunoreactive terminals and processes. Only its more densely cellular ventrolateral portion contained appreciable fiber and terminal staining. One of the more distinctive patterns of ChAT immunoreactivity was seen in the nucleus of the lateral olfactory tract. Here, ChAT positive fibers formed pericellular basket plexuses around unstained cell bodies. This unique pattern of staining was used to delineate the boundaries of the nucleus and indicated that it is present for much of the rostrocaudal extent of the amygdala. Another region of conspicuous staining on the medial surface of the amygdala was the sulcal portion of the periamygdaloid cortex. This region, associated with the sulcus semiannularis and bordering the entorhinal cortex, consistently contained dense immunoreactivity. The central nucleus also presented a somewhat idiosyncratic pattern of ChAT staining. The lateral subdivision had a diffuse distribution of immunoreactivity in which focal patches of more densely stained terminals and occasional fine fibers were embedded. In contrast, the medial subdivision contained a larger number of thicker, stained fibers without diffuse background labeling.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intrinsic connections of the rat amygdaloid complex: Projections originating in the accessory basal nucleus

The amygdaloid complex plays an important role in the detection of emotional stimuli, the generation of emotional responses, the formation of emotional memories, and perhaps other complex associational processes. These functions depend upon the flow of information through intricate and poorly understood circuitries within the amygdala. As part of an ongoing project aimed at further elucidating these circuits, we examined the intra-amygdaloid connections of the acessory basal nucleus in the rat. In addition, we examined connections of the anterior cortical nucleus and amygdalahippocampal area to determine whether portions of these nuclei should be included in the accessory basal nucleus (as some earlier studies suggest). Phaseolus vulgaris leucoagglutinin was injected into different rostrocaudal levels of the accessory basal nucleus (n = 12) or into the anterior cortical nucleus (n = 3) or amygdalahippocampal area (n = 2). The major intra-amygdaloid projections from the accessory basal nucleus were directed to the medial and capsular divisions of the central nucleus, the medial division of the amygdalohippocampal area, the medial division of the lateral nucleus, the central division of the medial nucleus, and the posterior cortical nucleus. The projections originating in the anterior cortical nucleus and the lateral division of the amygdalohippocampal area differed from those originating in the accessory basal nucleus, which suggests that these areas are not part of the deep amygdaloid nuclei have different intra-amygdaloid connections. The pattern of these various connections suggests that information entering the amygdala from different sources can be integrated only in certain amygdaloid regions. © 1996 Wiley-Liss, Inc.     

Topographic relations of cholinergic and noradrenergic neurons in the feline pontomesencephalic tegmentum: an immunohistochemical study

The immunohistochemical localization of the neurotransmitter synthesizing enzymes choline acetyltransferase, tyrosine hydroxylase and dopamine-beta-hydroxylase was examined in the feline pontomesencephalic tegmentum. Examination of adjacent sections stained for either choline acetyltransferase, tyrosine hydroxylase or dopamine-beta-hydroxylase immunoreactivity, as well as individual sections doubly stained for both choline acetyltransferase and tyrosine hydroxylase immunoreactivity, unequivocally demonstrated that noradrenergic and cholinergic neurons were extensively intermingled in the brainstem tegmentum of the cat. This contrasts with the situation in various other species, where neurons utilizing these two neurotransmitters are discretely localized in distinct nuclei. Furthermore, the present studies demonstrate the existence of two types of choline acetyltransferase immunoreactive neurons in the feline tegmentum: the magnocellular neurons of the pedunculopontine and laterodorsal tegmental nuclei which stain histochemically for NADPH diaphorase, plus a population of small spindle-shaped neurons in the medial and lateral parabrachial nuclei which do not stain positively for NADPH diaphorase. The data are discussed with respect to several influential hypotheses of sleep cycle control.