Cholinergic neuronal loss in the globus pallidus of Alzheimer disease patients.

Cholinergic neurons of the ventral pallidum and the dorsal pallidum (globus pallidus) were immunohistochemically investigated in patients suffering from Alzheimer disease (AD). Measurement of cholinergic neurons, stained with an antiserum against choline acetyltransferase (ChAT), revealed that their number was significantly reduced in both the dorsal pallidum (37.5%) and the ventral pallidum (65%) of AD patients (n = 4) when compared to control subjects (n = 3). No shrinkage of these cells was observed. The number of immunostained neuropeptide Y-containing neurons in the same structures was not different in controls and AD patients, indicating that the loss of cholinergic neurons was selective. These results combined with previous reports give further information upon which specific subsets of cholinergic neurons degenerate in AD.     

Abeta-degrading endopeptidase,neprilysin, in mouse brain: synaptic and axonal localization inversely correlating with Abeta pathology

Metabolism of amyloid-beta peptide (Abeta) is closely associated with the pathology and etiology of Alzheimer's disease (AD). Since neprilysin is the only rate-limiting catabolic peptidase proven by reverse genetics to participate in Abeta metabolism in vivo, we performed detailed immunohistochemical analysis of neprilysin in mouse brain using neprilysin-deficient mice as a negative control. The aim was to assess, at both the cellular and subcellular levels, where Abeta undergoes neprilysin-dependent degradation in the brain and how neprilysin localization relates to Abeta pathology in amyloid precursor protein (APP)-transgenic mice. In hippocampus, neprilysin was present in the stratum pyramidale and stratum lacunosum-moleculare of the CA1-3 fields and the molecular layer of the dentate gyrus. Confocal double immunofluorescence analyses revealed the subcellular localization of neprilysin along axons and at synapses. This observation suggests that after synthesis in the soma, neprilysin, a type II membrane-associated protein, is axonally transported to the terminals, where Abeta degradation is likely to take place. Among various cell types, GABAergic and metabotropic glutamate 2/3 receptor-positive neurons but not catecholaminergic or cholinergic neurons, expressed neprilysin in hippocampus and neocortex, implying the presence of a cell type-specific mechanism that regulates neprilysin gene expression. As expected, Abeta deposition correlated inversely with neprilysin expression in TgCRND8 APP-transgenic mice. These observations not only support the notion that neprilysin functions as a major Abeta-degrading enzyme in the brain but also suggest that down-regulation of neprilysin activity, which may be caused by aging, is likely to elevate local concentrations of Abeta at and around neuronal synapses.     

Convergent cholinergic activities in aging and Alzheimer's disease.

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities have been examined postmortem in a series of 66 individuals with no evidence of CNS disease, ranging in age from 24 gestational weeks to 95 years and in 33 cases of Alzheimer's disease (AD) aged 57-89 years. In the normal human hippocampus a striking and highly significant age-related decline in ChAT occurred from middle to old age (between 40 and 100 years); a trend apparent at a later stage and to a lesser extent in the hippocampal gyrus. In both areas enzyme activity in AD was inversely related to age at death; reductions compared with the normal were on average 70-80% in the 60-70 year old groups compared with 30-40% in the 80-90 year old group. A similar trend was apparent with respect to acetylcholinesterase (AchE) histochemical activity associated with fibers and terminals (predominantly cholinergic and concentrated in CA3 and 4 of the hippocampus) but not with reactive perikarya (considered to be noncholinergic) present in both hippocampus and cortex. These data indicate that the normal aging human hippocampus may constitute a useful model for investigating the dysfunction or degeneration of basal forebrain cholinergic neurons in AD.     

The superior olivary complex of the hamster has multiple periods of cholinergic neuron development

Cholinergic neurons of the superior olivary complex share a common embryological and phylogenetic origin with brainstem motor neurons and serve as the major descending efferent pathway either to the cochlea as part of the olivocochlear system or to the cochlear nucleus. In this study, we investigated the developmental expression patterns of choline acetyltransferase (ChAT) and its co-localization with calcitonin gene-related peptide within the superior olivary complex and neighboring brainstem motor nuclei. At embryonic day 12, neurons in the ventral nucleus of the trapezoid body were first to express ChAT. The temporal expression pattern of both ChAT mRNA and immunoreactivity in this periolivary region mimicked motor neurons in the facial and trigeminal motor nuclei. Just before birth, shell neurons surrounding the lateral superior olive expressed ChAT. Neither ChAT-positive periolivary neurons nor shell neurons co-expressed calcitonin gene-related peptide during development or in the adult. Immediately following birth, intrinsic neurons within the lateral superior olive expressed ChAT but not calcitonin gene-related peptide. However, a transient increase in the number of ChAT-positive neurons in the lateral superior olive coincided with the onset of the calcitonin gene-related peptide co-expression within these neurons. We conclude that ChAT expression appears first in periolivary regions containing medial olivocochlear neurons, precedes the expression of calcitonin gene-related peptide in the superior olivary complex, and is co-expressed with calcitonin gene-related peptide within the lateral superior olive containing lateral olivocochlear neurons. These data suggest that the lateral olivocochlear system co-expresses ChAT and calcitonin gene-related peptide, whereas the medial olivocochlear system does not.     

Expression and localization of pChAT as a novel method to study cholinergic innervation of rat adrenal gland

Cholinergic innervation of the rat adrenal gland has been analyzed previously using cholinergic markers including acetylcholinesterase (AChE), choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). In the present study, we demonstrate putative cholinergic neurons in the rat adrenal gland using an antibody to pChAT, which is the product of a splice variant of ChAT mRNA that is preferentially localized in peripheral cholinergic nerves. Most of the ganglionic neurons as well as small single sporadic neurons in the adrenal gland were stained intensely for pChAT. The density of pChAT-immunoreactive (IR) fibers was distinct in the adrenal cortex and medulla. AChE-, cChAT- and VAChT-immunoreactivities were also observed in some cells and fibers of the adrenal medulla, while the cortex had few positive nerve fibers. These results indicate that ganglionic neurons of the adrenal medulla and nerve fibers heterogeneously express cholinergic markers, especially pChAT. Furthermore, the innervation of the adrenal gland, cortex and medulla, by some cholinergic fibers provides additional morphological evidence for a significant role of cholinergic mechanisms in adrenal gland functions.     

Distribution of choline acetyltransferase immunoreactive somata in the feline brainstem: implications for REM sleep generation

In the present study we examined the distribution of cholinergic and catecholaminergic neurons, in the feline brainstem, as defined by choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) immunohistochemistry. In the dorsal tegmentum, ChAT immunoreactive neurons were distributed in the parabrachial area [the pedunculopontine group (PPG)] and along the medial adjacent central gray [the lateral dorsal tegmental group (LDT)]. The cholinergic neurons in the LDT area were larger than those in the PPG. When adjacent tissue sections were labeled with TH we noted extensive overlap between catecholamine and cholinergic neurons in the PPG, suggesting that REM sleep may occur as a result of an interaction between these transmitters in this area rather than the medial pontine reticular formation where no cholinergic or catecholamine neurons were found. Cholinergic neurons were also found in the cranial nerve nuclei and the nucleus ambiguus. The presence of cholinergic neurons in the PPG and LDT suggest that these neurons may play an important role in the generation of some of the tonic and phasic components of REM sleep, such as cortical desynchronization, pontogeniculo occipital waves, and muscle atonia.     

Projections from the rat pedunculopontine and laterodorsal tegmental nuclei to the anterior thalamus and ventral tegmental area arise from largely separate populations of neurons

Cholinergic and non-cholinergic neurons in the brainstem pedunculopontine (PPT) and laterodorsal tegmental (LDT) nuclei innervate diverse forebrain structures. The cholinergic neurons within these regions send heavy projections to thalamic nuclei and provide modulatory input as well to midbrain dopamine cells in the ventral tegmental area (VTA). Cholinergic PPT/LDT neurons are known to send collateralized projections to thalamic and non-thalamic targets, and previous studies have shown that many of the afferents to the VTA arise from neurons that also project to midline and intralaminar thalamic nuclei. However, whether cholinergic projections to the VTA and anterior thalamus (AT) are similarly collateralized is unknown. Ultrastructural work from our laboratory has demonstrated that cholinergic axon varicosities in these regions differ both morphologically and with respect to the expression and localization of the high-affinity choline transporter. We therefore hypothesized that the cholinergic innervation to these regions is provided by separate sets of PPT/LDT neurons. Dual retrograde tract-tracing from the AT and VTA indicated that only a small percentage of the total afferent population to either region showed evidence of providing collateralized input to the other target. Cholinergic and non-cholinergic cells displayed a similarly low percentage of collateralization. These results are contrasted to a control case in which retrograde labeling from the midline paratenial thalamic nucleus and the VTA resulted in higher percentages of cholinergic and non-cholinergic dual-tracer labeled cells. Our results indicate that functionally distinct limbic target regions receive primarily segregated signaling from PPT/LDT neurons.     

Genetic polymorphism in the cathepsin G gene and the risk of Alzheimer's disease

Cholinergic neurons in the laterodorsal tegmental nucleus (LDT) have important roles in the regulation of sleep or waking in adult animals. In neonatal animals, sleep is largely occupied by paradoxical sleep. To investigate the relation between the cholinergic neurons in the LDT and the development of neonatal sleep, we dissected the LDT of rat by micropunch method at postnatal day 1--45 and measured the activity of choline acetyltransferase (ChAT). Either specific or total activity of ChAT was weak in the first week, increased strikingly in the second week and then moderately thereafter. The time course of the increase in ChAT activity correlates well to that of the decrease in the amount of paradoxical sleep or body twitches after birth.     

Improvement of cognitive function and physical activity of aging mice by human neural stem cells over-expressing choline acetyltransferase

Aging is characterized by progressive loss of cognitive and memory functions as well as decrease in physical activities. In the present study, a human neural stem cell line (F3 NSC) over-expressing choline acetyltransferase (F3.ChAT), an enzyme responsible for acetylcholine synthesis, was generated and transplanted in the brain of 18-month-old male ICR mice. Four weeks post-transplantation, neurobehavioral functions, expression of ChAT enzyme, production of acetylcholine and neurotrophic factors, and expression of cholinergic nervous system markers in transplanted animals were investigated. F3.ChAT NSCs markedly improved both the cognitive function and physical activity of aging animals, in parallel with the elevation of brain acetylcholine level. Transplanted F3 and F3.ChAT cells were found to differentiate into neurons and astrocytes, and to produce ChAT proteins. Transplantation of the stem cells increased brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), enhanced expression of Trk B, and restored host microtubule-associated protein 2 and cholinergic nervous system. The results demonstrate that human NSCs over-expressing ChAT improve cognitive function and physical activity of aging mice, not only by producing ACh directly but also by restoring cholinergic neuronal integrity, which might be mediated by neurotrophins BDNF and NGF.     

Cholinergic innervation and thalamic input in rat nucleus accumbens

Cholinergic interneurons are the only known source of acetylcholine in the rat nucleus accumbens (nAcb); yet there is little anatomical data about their mode of innervation and the origin of their excitatory drive. We characterized the cholinergic and thalamic innervations of nAcb with choline acetyltransferase (ChAT) immunocytochemistry and anterograde transport of Phaseolus vulgaris-leucoagglutinin (PHA-L) from the midline/intralaminar/paraventricular thalamic nuclei. The use of a monoclonal ChAT antiserum against whole rat ChAT protein allowed for an optimal visualization of the small dendritic branches and fine varicose axons of cholinergic interneurons. PHA-L-labeled thalamic afferents were heterogeneously distributed throughout the core and shell regions of nAcb, overlapping regionally with cholinergic somata and dendrites. At the ultrastructural level, several hundred single-section profiles of PHA-L and ChAT-labeled axon terminals were analyzed for morphology, synaptic frequency, and the nature of their synaptic targets. The cholinergic profiles were small and apposed to various neuronal elements, but rarely exhibited a synaptic membrane specialization (5% in single ultrathin sections). Stereological extrapolation indicated that less than 15% of these cholinergic varicosities were synaptic. The PHA-L-labeled profiles were comparatively large and often synaptic (37% in single ultrathin sections), making asymmetrical contacts primarily with dendritic spines (>90%). Stereological extrapolation indicated that all PHA-L-labeled terminals were synaptic. In double-labeled material, some PHA-L-labeled terminals were directly apposed to ChAT-labeled somata or dendrites, but synapses were never seen between the two types of elements. These observations demonstrate that the cholinergic innervation of rat nAcb is largely asynaptic. They confirm that the afferents from midline/intralaminar/paraventricular thalamic nuclei to rat nAcb synapse mostly on dendritic spines, presumably of medium spiny neurons, and suggest that the excitatory drive of nAcb cholinergic interneurons from thalamus is indirect, either via substance P release from recurrent collaterals of medium spiny neurons and/or by extrasynaptic diffusion of glutamate.     

Neocortical morphometry and cholinergic neurochemistry in Pick''s disease.

With a computerized image-analysis apparatus for neocortical morphometry and chemical methods for evaluation of the cholinergic system, five brain specimens of Pick's disease (PD) were studied and the results compared to those from specimens of age-matched normal subjects and Alzheimer's disease (AD). The PD specimens showed major reductions in brain weight, frontal and temporal cortical thickness, and large neuron populations, compared with controls. Lesser reductions were seen in small neurons and thickness of the inferior parietal cortex. The authors found no relationship between age of onset or disease duration and either the degree of cortical thinning or neuron loss or the number of Pick bodies in the neocortex and hippocampus. PD specimens were more atrophic than AD brains, having lower brain weights and more fronto-temporal thinning. Large neurons were comparably reduced in the two conditions in the frontal and temporal lobes, but small neuron losses were greater in the PD midfrontal area. Only the AD cases showed loss of large neurons in the inferior parietal region. Levels of choline acetyltransferase were normal in PD and reduced in AD, whereas muscarinic receptor binding was decreased in both.     

Human cholinergic basal forebrain: chemoanatomy and neurologic dysfunction

The human cholinergic basal forebrain (CBF) is comprised of magnocellular hyperchromic neurons within the septal/diagonal band complex and nucleus basalis (NB) of Meynert. CBF neurons provide the major cholinergic innervation to the hippocampus, amygdala and neocortex. They play a role in cognition and attentional behaviors, and are dysfunctional in Alzheimer's disease (AD). The human CBF displays a continuum of large cells that contain various cholinergic markers, nerve growth factor (NGF) and its cognate receptors, calbindin, glutamate receptors, and the estrogen receptors, ER and ERβ. Admixed with these cholinergic neuronal phenotypes are smaller interneurons containing the m2 muscarinic acetylcholine receptor (mAChRs), NADPH-diaphorase, GABA, calcium binding proteins and several inhibitory neuropeptides including galanin (GAL), which is over expressed in AD. Studies using human autopsy material indicate an age-related dissociation of calbindin and the glutamate receptor GluR2 within CBF neurons, suggesting that these molecules act synergistically to induce excitotoxic cell death during aging, and possibly during AD. Choline acetyltrasnferease (ChAT) activity and CBF neuron number is preserved in the cholinergic basocortical system and up regulated in the septohippocampal system during prodromal as compared with end stage AD. In contrast, the number of CBF neurons containing NGF receptors is reduced early in the disease process suggesting a phenotypic silence and not a frank loss of neurons. In end stage AD, there is a selective reduction in trkA mRNA but not p75^NTR in single CBF cells suggesting a neurotrophic defect throughout the progression of AD. These observations indicate the complexity of the chemoanatomy of the human CBF and suggest that multiple factors play different roles in its dysfunction in aging and AD.     

Age-related changes in rostral basal forebrain cholinergic and GABAergic projection neurons: relationship with spatial impairment

Both cholinergic and GABAergic projections from the rostral basal forebrain contribute to hippocampal function and mnemonic abilities. While dysfunction of cholinergic neurons has been heavily implicated in age-related memory decline, significantly less is known regarding how age-related changes in codistributed GABAergic projection neurons contribute to a decline in hippocampal-dependent spatial learning. In the current study, confocal stereology was used to quantify cholinergic (choline acetyltransferase [ChAT] immunopositive) neurons, GABAergic projection (glutamic decarboxylase 67 [GAD67] immunopositive) neurons, and total (neuronal nuclei [NeuN] immunopositive) neurons in the rostral basal forebrain of young and aged rats that were first characterized on a spatial learning task. ChAT immunopositive neurons were significantly but modestly reduced in aged rats. Although ChAT immunopositive neuron number was strongly correlated with spatial learning abilities among young rats, the reduction of ChAT immunopositive neurons was not associated with impaired spatial learning in aged rats. In contrast, the number of GAD67 immunopositive neurons was robustly and selectively elevated in aged rats that exhibited impaired spatial learning. Interestingly, the total number of rostral basal forebrain neurons was comparable in young and aged rats, regardless of their cognitive status. These data demonstrate differential effects of age on phenotypically distinct rostral basal forebrain projection neurons, and implicate dysregulated cholinergic and GABAergic septohippocampal circuitry in age-related mnemonic decline.     

Immunohistochemical staining of cholinergic neurons in the human brain using a polyclonal antibody to human choline acetyltransferase

Antibodies against human placental choline acetyltransferase (ChAT) were used to immunohistochemically stain cholinergic neurons in the neostriatum and nucleus basalis of Meynert in human brain. Cells in both regions were intensely stained as were nerve fibers. Comparable cells were stained in these same brain regions in the rat. This anti-human ChAT antibody will enable the further detailed characterization of cholinergic neurons in the human brain in both health and disease.     

胰岛素基因增强结合蛋白1基因修饰的神经干细胞向胆碱能神经元分化的实验研究

目的 探讨大鼠胰岛素基因增强结合蛋白1(Islet-1)基因是否有诱导神经干细胞(NSCs)向胆碱能运动神经元分化的能力.方法 用Islet-1基因重组逆转录病毒载体转导NSCs后,用免疫荧光组织化学染色方法检测Islet-1在NSCs内的表达;体内、体外实验观察导入Islet-1基因的NSCs向乙酰胆碱转移酶(ChAT)阳性细胞分化的情况.结果 在体外分化实验中观察到,转导Islet-1基因的NSCs向胆碱能运动神经元分化的细胞数明显多于对照组;体内移植实验发现,导入Islet-1基因的NSCs在体内可以向ChAT阳性细胞分化.结论 Islet-1基因有诱导NSCs向胆碱能运动神经元分化的能力.     

Drain of the brain: low-affinity p75 neurotrophin receptor affords a molecular sink for clearance of cortical amyloid β by the cholinergic modulator system

Cholinergic basal forebrain (BF) neurons source one of the largest modulator systems of the brain, supplying acetylcholine to the entire cerebral mantle. Ample evidence suggests a causal link between the depletion of cortical acetylcholine and the selective disruption of cognitive functions in the course of aging and Alzheimer's disease (AD). A distinctive yet underappreciated feature of BF cholinergic neurons is their enrichment with the p75 neurotrophin receptor (p75^NTR), which is also recognized as a high-affinity acceptor for the amyloid-β (Aβ) peptide. Herein, we critically overview the emerging data, which suggest the relevance of p75^NTR-mediated uptake of Aβ followed by its degradation in lysosomes of BF cholinergic neurons for the homeostasis and clearance of this peptide from the cerebral cortex. We propose that via such a unique arrangement, cholinergic neurons afford their functional targets with an efficient molecular “drain” for Aβ. This process is suggested as the proximal cause for the greater “wear and tear” of the BF cholinergic system during aging and especially AD.     

Cholinergic neurons in the telencephalon of the reptile Caiman crocodilus

Choline acetyltransferase (ChAT) immunohistochemistry was used to characterize the distribution of cholinergic neurons in the telencephalon of the reptile, Caiman crocodilus. ChAT-positive cell bodies were observed within the small-celled portion of the ventrolateral area of the telencephalon (VLA s.c.), a region previously considered comparable to the mammalian caudate nucleus and putamen. A large field of cholinergic neurons was observed within the ventral paleostriatum (VP), a region comparable to the substantia innominata and ventral pallidum of mammals. Cholinergic neurons were also observed within cranial motor nuclei of the brainstem, within the isthmic nucleus and within portions of the lateral reticular formation of the pons and medulla. A rich plexus of cholinergic fibers was observed within the intermediate and deep layers of the optic tectum. The results of this study indicate that many aspects of cholinergic system organization are similar in caiman and mammals, and suggest a common derivation of these systems from ancestral forms.