Heterogeneity and selectivity of the degeneration of cholinergic neurons in the basal forebrain of patients with Alzheimer's disease

Cholinergic neurons were studied by immunohistochemistry, with an antiserum against choline acetyltransferase (ChAT), in the basal forebrain (Ch1 to Ch4) of four patients with Alzheimer's disease (AD) and four control subjects. ChAT-positive cell bodies were mapped and counted in Ch1 (medial septal nucleus), Ch2 (vertical nucleus of the diagonal band), Ch3 (horizontal nucleus of the diagonal band) and Ch4 (nucleus basalis of Meynert). Compared to controls, the number of cholinergic neurons in AD patients was reduced by 50% on average. The interindividual variations in cholinergic cell loss were high, neuronal loss ranging from moderate (27%) to severe (63%). Despite the small number of brains studied, a significant correlation was found between the cholinergic cell loss and the degree of intellectual impairment. To determine the selectivity of cholinergic neuronal loss in the basal forebrain of AD patients, NPY-immunoreactive neurons were also investigated. The number of NPY-positive cell bodies was the same in controls and AD patients. The results (1) confirm cholinergic neuron degeneration in the basal forebrain in AD and the relative sparing of these neurons in some patients, (2) indicate that degneration of cholinergic neurons in the basal forebrain contributes to intellectual decline, and (3) show that, in AD, such cholinergic cell loss is selective, since NPY-positive neurons are preserved in the basal forebrain.     

Nerve growth factor receptor immunoreactivity within the nucleus basalis (Ch4) in Parkinson's disease: reduced cell numbers and co-localization with cholinergic neurons.

In a effort to better define the role cholinergic basal forebrain neurons play in human cognitive processes, a quantitative assessment of cholinergic nucleus basalis (Ch4) neurons was carried out in 5 patients with Parkinson's disease (PD; 4 non-demented and 1 demented) and 4 age-matched controls using nerve growth factor (NGF) receptor immunohistochemistry as a direct marker for cholinergic basal forebrain neurons. Virtually all (greater than 90%) NGF receptor-containing neurons co-localize with the specific cholinergic marker choline acetyltransferase (ChAT) within the nucleus basalis in PD. NGF receptor-containing neurons were reduced on average by 68% (range 38.6-87.4%) in the non-demented PD cases and by 88.6% in the demented PD patient. Loss of these neurons was heterogeneous across the nucleus basalis subfields with only the anterolateral and posterior Ch4 subregions demonstrating significant reductions of NGF receptor-containing neurons. The reduction in NGF receptor-containing neurons was accompanied by a decrease of acetylcholinesterase (AChE) containing fibers within temporal cortex and in some cases ChAT immunoreactivity in the basolateral amygdaloid nucleus. The numerous non-cholinergic AChE-rich pyramidal cells which were observed throughout the cortex of aged controls were also virtually absent in PD. Although PD patients exhibited severe reductions in Ch4 neurons, few neuritic plaques or neurofibrillary tangles were observed within the PD cortex or Ch4 and similar numbers of these AD-type pathologies were seen within age-matched controls. This suggests that Ch4 degeneration alone is not sufficient to induce such cytoskeletal abnormalities and that the neuron loss seen within Ch4 in AD and PD may be mediated through different processes. These results, coupled with the extensive basic and clinical literature linking acetylcholine and memory function, further indicate that Ch4 degeneration without additional cortical and/or subcortical pathology is not sufficient to impair cognition in PD. Perhaps additional pathology must be superimposed upon nucleus basalis degeneration to induce dementia in humans.     

The nucleus basalis in Huntington's disease

The nucleus basalis of Meynert (nbM) provides most of the cholinergic input to the cerebral cortex. The loss of cortical choline acetyltransferase (CAT) activity in Alzheimer's disease (AD) and senile dementia of the Alzheimer's type (SDAT) appears to be related to a severe depopulation of the nbM in this dementia. In Huntington's disease (HD), by contrast, there is no loss of cortical CAT activity. The present quantitative study indicates that (1) there is no significant loss of neurons from the nbM in HD, and (2) that the previously described cytologic changes in the neurons of this nucleus in HD patients do not differ significantly from controls. These findings are consistent with the working hypothesis that the types of dementia associated with reductions of neocortical CAT activity are characterized by dysfunction or death of neurons in the nbM, but dementing disorders with normal neocortical CAT activity manifest no major abnormalities in this cholinergic nucleus of the basal forebrain.     

Galanin fiber hypertrophy within the cholinergic nucleus basalis during the progression of Alzheimer's disease

Galanin (GAL)-containing fibers enlarge and hyperinnervate remaining cholinergic basal forebrain (CBF) neurons within the anterior nucleus basalis (NB) in late-stage Alzheimer's disease (AD). Whether GAL hypertrophy occurs in the CBF in the prodromal or early stages of AD remains unknown. The present study used GAL immunohistochemistry and an unbiased semiquantitative scoring method to evaluate GAL innervation in the anterior NB of subjects clinically diagnosed as having no cognitive impairment, mild cognitive impairment or early-stage (mild/moderate) AD. There was no difference in GAL fiber staining within the anterior NB across the three clinical groups examined. Furthermore, GAL fiber innervation was not correlated with the number of NB neurons expressing the nerve growth factor receptors p75(NTR) or TrkA or with cortical choline acetyltransferase activity in the same cases. Single-cell gene expression analysis demonstrated that cholinergic NB neurons express mRNA for the GAL receptors GALR1, GALR2 and GALR3, yet the levels of these mRNAs were unchanged across the three diagnostic groups. These observations indicate that GAL hypertrophy within the anterior NB subfield is a late-stage AD response, which may play a role in regulating the cholinergic tone of remaining basocortical projection neurons.     

TrkA gene ablation in basal forebrain results in dysfunction of the cholinergic circuitry

Dysfunction of basal forebrain cholinergic neurons (BFCNs) is an early pathological hallmark of Alzheimer's disease (AD). Numerous studies have indicated that nerve growth factor (NGF) supports survival and phenotypic differentiation of BFCNs. Consistent with a potential link to AD pathogenesis, TrkA, a NGF receptor, is expressed in cholinergic forebrain neuronal populations including those in BF and striatum, and is markedly reduced in individuals with mild cognitive impairment (MCI) without dementia and early-stage AD. To investigate the role of TrkA in the development, connectivity, and function of the BF cholinergic system and its contribution to AD pathology, we have generated a forebrain-specific conditional TrkA knock-out mouse line. Our findings show a key role for TrkA signaling in establishing the BF cholinergic circuitry through the ERK pathway, and demonstrate that the normal developmental increase of choline acetyltransferase expression becomes critically dependent on TrkA signaling before neuronal connections are established. Moreover, the anatomical and physiological deficits caused by lack of TrkA signaling in BFCNs have selective impact on cognitive activity. These data demonstrate that TrkA loss results in cholinergic BF dysfunction and cognitive decline that is reminiscent of MCI and early AD.     

Basal forebrain and mesopontine tegmental projections to the reticular thalamic nucleus: an axonal collateralization and immunohistochemical study in the rat

Using a double fluorescence retrograde labeling procedure, the present study sought to determine the degree to which basal forebrain and mesopontine tegmental neurons have axons that innervate both the reticular thalamic nucleus and the cerebral cortex. Immunofluorescence for choline acetyltransferase, somatostatin, and the calcium-binding protein parvalbumin was also performed to elucidate the neurochemical identity of basal forebrain and mesopontine tegmental inputs to the reticular thalamic nucleus. A significant portion (10-15%) of neurons in the basal forebrain and mesopontine tegmentum that were retrogradely labeled from the reticular thalamic nucleus were also found to be retrogradely labeled from the cortex. Many of these neurons stained positively for choline acetyltransferase. Of the basal forebrain neurons retrogradely labeled from the reticular thalamic nucleus, approximately 20% were found to be immunoreactive to choline acetyltransferase, whereas none was stained for somatostatin. A larger portion (up to 50%) of the basal forebrain neurons that were retrogradely labeled from the reticular thalamic nucleus were parvalbumin-immunoreactive, and some of these were also retrogradely labeled from the cortex. These results suggest that a subpopulation of cholinergic and non-cholinergic neurons in the basal forebrain and the mesopontine tegmentum may influence simultaneously the activity of neurons in the reticular thalamic nucleus and the cerebral cortex.     

Cholinergic forebrain degeneration in the APPswe/PS1DeltaE9 transgenic mouse

The impact of Abeta deposition upon cholinergic intrinsic cortical and striatal, as well as basal forebrain long projection neuronal systems was qualitatively and quantitatively evaluated in young (2-6 months) and middle-aged (10-16 months) APPswe/PS1DeltaE9 transgenic (tg) mice. Cholinergic neuritic swellings occurred as early as 2-3 months of age in the cortex and hippocampus and 5-6 months in the striatum of tg mice. However, cholinergic neuron number or choline acetyltransferase (ChAT) optical density measurements remained unchanged in the forebrain structures with age in APPswe/PS1DeltaE9 tg mice. ChAT enzyme activity decreased significantly in the cortex and hippocampus of middle-aged tg mice. These results suggest that Abeta deposition has age-dependent effects on cortical and hippocampal ChAT fiber networks and enzyme activity, but does not impact the survival of cholinergic intrinsic or long projection forebrain neurons in APPswe/PS1DeltaE9 tg mice.     

GABAergic basal forebrain neurons that express receptor for neurokinin B and send axons to the cerebral cortex

Neurons expressing neurokinin B (NK3) receptor in the basal forebrain region of rats were characterized histochemically by combining immunocytochemistry, in situ hybridization and retrograde labeling, and electrophysiologically by whole-cell clamp recording. NK3 receptor-immunoreactive neurons were found in the basal forebrain region including the substantia innominata, where axon terminals immunoreactive for preprotachykinin B, the precursor peptide of neurokinin B (NKB), were densely distributed. More than 90% of NK3 receptor-expressing neurons in the basal forebrain region showed signals for glutamate decarboxylase mRNA, indicating that almost all NK3 receptor-expressing neurons were gamma-aminobutyric acid (GABA)ergic neurons. On the other hand, only a few NK3 receptor-immunoreactive neurons showed immunoreactivity for choline acetyltransferase or parvalbumin in the substantia innominata, ventral pallidum, and globus pallidus, although the distribution of NK3 receptor-expressing neurons overlapped with those of cholinergic neurons and parvalbumin-positive neurons. After injection of wheat germ agglutinin into the cerebral cortex, NK3 receptor immunoreactivity was detected in about 25% of retrogradely labeled basal forebrain neurons, indicating that NK3 receptor-expressing neurons send projection fibers to the cerebral cortex. In the whole-cell clamp recording study, a selective NK3 receptor agonist evoked membrane depolarization or inward currents with decrease of input impedance in 10 of 100 cortically projecting neurons recorded in the basal forebrain region. Because NKB-producing striatal neurons send axons selectively to the basal forebrain region, the present results suggest that the release of NKB by those striatal neurons induces an inhibitory effect on cortical neurons via facilitation of GABAergic basal forebrain neurons expressing NK3 receptor.     

The distribution pattern of pathology and cholinergic deficits in amygdaloid complex in Alzheimer's disease and dementia with Lewy bodies

We studied the distribution pattern of pathology and cholinergic deficits in the subnuclei of the amygdaloid complex (AC) in five patients with Alzheimer's disease (AD), eight with dementia with Lewy bodies (DLB) and five normal controls. In controls, the basal nucleus contained the highest choline acetyltransferase activity; the activity in the lateral and central nuclei and those in the cortical, medial and accessory basal nuclei were comparable. In AD, there was a significant decrease in choline acetyltransferase activity in the accessory basal and lateral nuclei, in DLB a significant decrease was observed in the accessory basal, lateral and cortical nuclei. Compared to controls the hyperphosphorylated tau-pathology burden was significantly higher in the basal, central and medial nuclei in AD and in the central, cortical, lateral and medial nuclei in DLB. The amyloid plaque burden was significantly higher in the accessory basal, basal, lateral and cortical nuclei in AD and in all nuclei in DLB. The α-synuclein burden was significantly higher in all nuclei in both AD and DLB. Compared to AD α-synuclein burden was higher in all nuclei in DLB. There were no correlations between the distribution pattern of hyperphosphorylated tau-pathology, amyloid plaques and α-synuclein-positive structures, and choline acetyltransferase activity, except the lateral nucleus in DLB. In conclusion we found no relationship between the pattern of cholinergic deficits and the distribution pattern of lesions in the AC of patients with AD or DLB. Cholinergic deficits were more prominent in the nuclei of basolateral (BL) group in AD, whereas the nuclei of both BL and corticomedial groups were involved in DLB, which may be due to the involvement of both basal forebrain and brainstem cholinergic nuclei in the latter.     

The basal forebrain cholinergic system in aging and dementia. Rescuing cholinergic neurons from neurotoxic amyloid-β42 with memantine

The dysfunction and loss of basal forebrain cholinergic neurons and their cortical projections are among the earliest pathological events in the pathogenesis of Alzheimer's disease (AD). The evidence pointing to cholinergic impairments come from studies that report a decline in the activity of choline acetyltransferase (ChAT) and acetylcholine esterase (AChE), acetylcholine (ACh) release and the levels of nicotinic and muscarinic receptors, and loss of cholinergic basal forebrain neurons in the AD brain. Alzheimer's disease pathology is characterized by an extensive loss of synapses and neuritic branchings which are the dominant scenario as compared to the loss of the neuronal cell bodies themselves. The appearance of cholinergic neuritic dystrophy, i.e. aberrant fibers and fiber swelling are more and more pronounced during brain aging and widely common in AD. When taking amyloid-β (Aβ) deposition as the ultimate causal factor of Alzheimer's disease the role of Aβ in cholinergic dysfunction should be considered. In that respect it has been stated that ACh release and synthesis are depressed, axonal transport is inhibited, and that ACh degradation is affected in the presence of Aβ peptides. β-Amyloid peptide 1-42, the principal constituent of the neuritic plaques seen in AD patients, is known to trigger excess amount of glutamate in the synaptic cleft by inhibiting the astroglial glutamate transporter and to increase the intracellular Ca²⁺ level. Based on the glutamatergic overexcitation theory of AD progression, the function of NMDA receptors and treatment with NMDA antagonists underlie some recent therapeutic applications. Memantine, a moderate affinity uncompetitive NMDA receptor antagonist interacts with its target only during states of pathological activation but does not interfere with the physiological receptor functions. In this study the neuroprotective effect of memantine on the forebrain cholinergic neurons against Aβ42 oligomers-induced toxicity was studied in an in vivo rat dementia model. We found that memantine rescued the neocortical cholinergic fibers originating from the basal forebrain cholinergic neurons, attenuated microglial activation around the intracerebral lesion sides, and improved attention and memory of Aβ42-injected rats exhibiting impaired learning and loss of cholinergic innervation of neocortex.     

Preservation of nucleus basalis neurons containing choline acetyltransferase and the vesicular acetylcholine transporter in the elderly with mild cognitive impairment and early Alzheimer's disease.

Immunocytochemistry for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) was used to examine the expression of these linked cholinergic markers in human basal forebrain, including cases with early stages of Alzheimer's disease (AD). Previous neurochemical studies have measured decreased ChAT activity in terminal fields, but little change or even increased levels of VAChT. To determine total cholinergic neuron numbers in the nucleus basalis of Meynert (nbM), stereologic methods were applied to tissue derived from three groups of individuals with varying levels of cognition: no cognitive impairment (NCI), mild cognitive impairment (MCI), and early-stage Alzheimer's disease (AD). Both markers were expressed robustly in nucleus basalis neurons and across all three groups. On average, there was no significant difference between the number of ChAT- (210,000) and VAChT- (174, 000) immunopositive neurons in the nbM per hemisphere in NCI cases for which the biological variation was calculated to be 17%. There was approximately a 15% nonsignificant reduction in the number of cholinergic neurons in the nbM in the AD cases with no decline in MCI cases. The number of ChAT- and VAChT-immunopositive neurons was shown to correlate significantly with the severity of dementia determined by scores on the Mini-Mental State Examination, but showed no relationship to apolipoprotein E allele status, age, gender, education, or postmortem interval when all clinical groups were combined or evaluated separately. These data suggest that cholinergic neurons, and the coexpression of ChAT and VAChT, are relatively preserved in early stages of AD.     

Development of AChE-positive neuronal projections from basal forebrain to cerebral cortex in organotypic tissue slice cultures.

Development of the innervation of the cerebral cortex by acetylcholinesterase (AChE)-stained basal forebrain neurons was studied in vitro using the roller tube technique. Slice cultures were maintained from 3 days to 4 weeks either in serum based medium or in chemically defined medium, each supplemented in some cases with nerve growth factor (NGF). The distribution of AChE and choline acetyltransferase (CAT)-containing neurons was investigated using histo- and immunocytochemical techniques. Slice cultures of basal forebrain revealed the presence of large and medium sized AChE-positive neurons. Within one week of cultivation, numerous AChE-labeled fibers could be seen growing out from the basal forebrain toward the cortex. After entering cortical tissue most of the afferent basal forebrain fibers projected either radially or obliquely into the cortical layers. Many afferent axons initially also travelled tangentially within the white matter, and turned then to grow into the cortical layers. Cerebral cortex tissue maintained a coarse laminar organization. Ramifications of basal forebrain fibers were visible within the subplate region, the deep and superficial cortical layers, and within the marginal zone; greatest density occurred in the subplate region and in marginal zones. Many of these processes exhibited branching patterns markedly similar to those observed during cortical development in vivo. Cortex slices placed with the pial surface adjacent to the basal forebrain revealed AChE-stained fibers that entered the cortical tissue through the marginal surface and gave off ramifications within the superficial layers and, less frequently, the deeper cortical layers. CAT-immunostaining revealed labeled cell bodies and neurites only in the basal forebrain, not in the cortex tissue. Control experiments with co-cultures of basal forebrain and cerebellum slices showed no AChE-positive fiber ingrowth into the cerebellum tissue. The results of these studies demonstrate that basal forebrain projections to cerebral cortex in vitro appear similar to the projections that develop in vivo, and indicate that organotypic co-cultures provide a valuable model for studies of developing cortical afferents.     

Fiber pathways of basal forebrain cholinergic neurons in monkeys

In rhesus monkeys, autoradiographic tracing methods, complemented by immunocytochemical and histochemical techniques, were used to delineate pathways by which cholinergic neurons of the nucleus basalis of Meynert (nbM) and nucleus of the diagonal band of Broca (ndbB) project to forebrain targets. Following injections of [3H]amino acids into these nuclei, 5 major fiber pathways were identified: axons of the nbM and ndbB project medially, principally within the cingulum bundle, to dorsomedial portions of the hemispheres; nbM and ndbB fibers exit laterally beneath the pallidum and striatum, enter the external and extreme capsules, and pass within the corona radiata to terminate in lateral and caudal regions of neocortex; axons coursing ventrally from the nbM project to portions of the temporal lobe, including the amygdala; some fibers pass through the fibrae pass orbitofrontales to the orbitofrontal cortex; and, finally axons of the nbM/ndbB project via the fimbria/rornix and a ventral pathway to the hippocampus. The presence of these 5 radiolabeled pathways arising from basal forebrain cholinergic neurons was confirmed by acetylcholinesterase histochemistry and choline acetyltransferase immunocytochemistry.     

Reductions in acetylcholine and nicotine binding in several degenerative diseases

Alzheimer's disease, Parkinson's disease, and progressive supranuclear palsy are all characterized by loss of neurons in the basal forebrain cholinergic system and by associated reductions in cortical presynaptic cholinergic markers, such as choline acetyltransferase. In this report, we identify that a major cortical receptor alteration in these disorders is a reduction in nicotinic receptors measured using both tritiated acetylcholine and levorotatory tritiated nicotine binding.     

Choline acetyltransferase activity associated with cerebral cortical microvessels does not originate in basal forebrain neurons

Cerebral cortical microvessels are innervated by cholinergic fibers that are probably involved in the regulation of local cerebral blood flow and blood-brain barrier permeability. The possibility exists that the cholinergic terminals associated with the cortical microvasculature belong to neurons from the nucleus basalis magnocellularis (NBM), where 70% of the cortical cholinergic projections originate. To test this hypothesis, ibotenic acid (25 nmol) was injected unilaterally in the NBM in rats, and 14 days later, choline acetyltransferase (ChAT) activity was measured in the frontoparietal cortex and in a blood vessel fraction isolated from this region. Lesions of the NBM resulted in a 50% decrease of cortical ChAT as compared with control or sham-operated hemispheres; however, no changes were observed in the ChAT activity associated with cortical microvessels. These results indicate that, in rat cerebral cortex, the perivascular cholinergic terminals do not originate in the basal forebrain.     

Effects of estrogen replacement therapy on cholinergic basal forebrain neurons and cortical cholinergic innervation in young and aged ovariectomized rhesus monkeys

Estrogen modulates the function of cholinergic basal forebrain neurons in aged female rats. The present study tested the hypothesis that estrogen enhances the phenotype of cholinergic basal forebrain neurons and their cortical cholinergic innervation in young adult and aged ovariectomized rhesus monkeys. Sixteen monkeys (9 young and 7 aged) received two injections of estradiol cypionate or vehicle separated by 3 weeks. All monkeys were killed 1 day after the last injection. Quantitative immunofluorescence in the vertical limb of the diagonal band (VLDB) revealed enhanced optical density for choline acetyltransferase (ChAT) in both young and aged monkeys treated with estrogen. In contrast, optical density for low-affinity p75 neurotrophin receptor immunoreactivity in the VLDB did not change after estrogen treatment in either aged or young animals. Quantitative immunofluorescence for either ChAT or the low-affinity p75 neurotrophin receptor in the nucleus basalis Meynert failed to reveal differences between vehicle and estrogen treatment in either age group. Quantitative estimates of acetylcholinesterase (AChE) fiber density revealed that estrogen-treated aged monkeys but not their younger counterparts had decreased numbers of AChE-positive fibers in layer II of frontal, insular, and cingulate cortices. These data indicate that estrogen administered in a manner simulating natural hormonal cyclicity produces modest age-specific chemical phenotypic and regional changes in select neuronal subfields of the cholinergic basal forebrain and their cortical projection sites in nonhuman primates.     

Loss of basal forebrain P75(NTR) immunoreactivity in subjects with mild cognitive impairment and Alzheimer's disease.

The long-held belief that degeneration of the cholinergic basal forebrain was central to Alzheimer's disease (AD) pathogenesis and occurred early in the disease process has been questioned recently. In this regard, changes in some cholinergic basal forebrain (CBF) markers (e.g. the high affinity trkA receptor) but not others (e.g., cortical choline acetyltransferase [ChAT] activity, the number of ChAT and vesicular acetylcholine transporter-immunoreactive neurons) suggest specific phenotypic changes, but not frank neuronal degeneration, early in the disease process. The present study examined the expression of the low affinity p75 neurotrophin receptor (p75(NTR)), an excellent marker of CBF neurons, in postmortem tissue derived from clinically well-characterized individuals who have been classified as having no cognitive impairment (NCI), mild cognitive impairment (MCI), and mild AD. Relative to NCI individuals, a significant and similar reduction in the number of nucleus basalis p75(NTR)-immunoreactive neurons was seen in individuals with MCI (38%) and mild AD (43%). The number of p75(NTR)-immunoreactive nucleus basalis neurons was significantly correlated with performance on the Mini-Mental State Exam, a Global Cognitive Test score, as well as some individual tests of working memory and attention. These data, together with previous reports, support the concept that phenotypic changes, but not frank neuronal degeneration, occur early in cognitive decline. Although there was no difference in p75(NTR) CBF cell reduction between MCI and AD, it remains to be determined whether these findings lend support to the hypothesis that MCI is a prodromal stage of AD.     

Comparison of nerve growth factor's effects on development of septum, striatum, and nucleus basalis cholinergic neurons in vitro

In the central nervous system, nerve growth factor (NGF) affects basal forebrain cholinergic neurons during early development and in the adult mammalian brain. These neurons are located in medial septum, diagonal band of Broca, and nucleus basalis of Meynert. While the effects of NGF on the development of septal cholinergic neurons are well documented, only little is known about the influence of NGF on development of cholinergic neurons in the nucleus basalis. In addition to the basal forebrain cholinergic neurons, there are cholinergic interneurons in the corpus striatum, which form an anatomically and functionally distinct population of cholinergic neurons. These striatal interneurons have been reported to respond to NGF during early development; however, it is not known whether the effects of NGF on their development are similar to those on septal cholinergic neurons. We prepared cultures of dissociated cells from fetal rat septum, striatum, and nucleus basalis and investigated the development of cholinergic neurons localized in these three different areas in the presence or absence of NGF. We now report that, first, cholinergic neurons of striatum and nucleus basalis develop a more extensive fiber network and contain more acetylcholinesterase (AChE) per neuron than do cholinergic neurons of septum. The amount of choline acetyltransferase (ChAT) per cholinergic neuron is approximately the same in all three culture types when grown in the absence of NGF. Second, NGF treatment increases and anti-NGF treatment decreases the number of AChE-positive neurons in cultures of low plating density, suggesting that NGF is able to promote survival of cholinergic neurons of all three areas studied. Third, NGF increases the total length of fibers and the number of branching points of cholinergic neurons in septal cultures but not in cultures of striatum and nucleus basalis. Fourth, NGF treatment increases AChE activity in septal but not in nucleus basalis or striatal cultures, suggesting that AChE activity reflects the extent of the fiber network of cholinergic neurons of all areas. Fifth, NGF treatment produces severalfold elevations in ChAT activity in septal cultures and more modest increases in cultures of nucleus basalis and striatum, suggesting that NGF is able to stimulate ChAT activity also in the absence of a stimulatory effect on survival and fiber growth. Our results demonstrate that, during early development, NGF is able to affect survival and differentiation of all three populations of forebrain cholinergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Apoptotic signals within the basal forebrain cholinergic neurons in Alzheimer's disease

A relatively early and substantial loss of basal forebrain cholinergic neurons is a constant feature of Alzheimer's disease (AD). However, the mechanisms that contribute to the selective vulnerability of these neurons are not fully delineated. In the present series of experiments, we determined the possible contribution of apoptotic processes and other pathologic cascades to the degeneration of the cholinergic neurons of the nucleus basalis of Meynert (NBM) in AD. In contrast to neurons in the frontal cortex which showed prominent DNA fragmentation as detected by the TUNEL method, no DNA fragmentation was observed within the NBM in any of the AD or normal brains. Similarly, immunoreactivity for the apoptotic signals Fas, Fas-ligand, Bax, Bcl-x, caspase-8, caspase-9 and caspase-3 was absent from the NBM of AD and control brains. In contrast, a substantial subpopulation of cholinergic neurons within the NBM in AD displayed prominent immunoreactivity for the apoptotic signal Fas-associated death domain (FADD) in the form of tangles. FADD immunoreactivity was also present in dystrophic neurites. FADD-positive tangle-like structures were localized in neurons which contained immunoreactivity for the cholinergic marker choline acetyltransferase (ChAT) and the low affinity neurotrophin receptor p75NTR. While many of the NBM cholinergic neurons in control brains contained immunoreactivity for the calcium binding protein calbindin-D28K (CB), the NBM neurons in AD displayed a substantial loss of CB immunoreactivity. Importantly, most of FADD-immunoreactive cholinergic neurons were devoid of CB immunoreactivity, and, conversely, most CB-positive cholinergic neurons had no FADD immunoreactivity. FADD immunoreactivity within the basal forebrain was colocalized with phosphorylated tau immunoreactive tangles and dystrophic neurites. In contrast, FADD immunoreactivity did not appear to be related to the primarily diffuse amyloid-beta deposits intermingled between cholinergic neurons in AD NBM. Finally, many CD68-positive microglia were observed surrounding the NBM cholinergic neurons in AD. In conclusion, the findings of the present study indicate that, while the FADD apoptotic signaling pathway may be triggered within the basal forebrain cholinergic neurons in AD, the apoptotic cascade is most likely aborted as no DNA fragmentation was detected and the executioner caspase-3 was not up-regulated within these neurons. The findings also suggest possible relationships between loss of CB, FADD expression and phosphorylation of tau within the basal forebrain cholinergic neurons in AD.