Progression of tau pathology in cholinergic Basal forebrain neurons in mild cognitive impairment and Alzheimer's disease

Tau is a microtubule-associated protein that forms neurofibrillary tangles (NFTs) in the selective vulnerable long projection neurons of the cholinergic basal forebrain (CBF) in Alzheimer's disease (AD). Although CBF neurodegeneration correlates with cognitive decline during AD progression, little is known about the temporal changes of tau accumulation in this region. We investigated tau posttranslational modifications during NFT evolution within the CBF neurons of the nucleus basalis (NB) using tissue from subjects with no cognitive impairment, mild cognitive impairment, and AD. The pS422 antibody was used as an early tau pathology marker that labels tau phosphorylated at Ser422; the TauC3 antibody was used to detect later stage tau pathology. Stereologic evaluation of NB tissue immunostained for pS422 and TauC3 revealed an increase in neurons expressing these tau epitopes during disease progression. We also investigated the occurrence of pretangle tau events within cholinergic NB neurons by dual staining for the cholinergic cell marker, p75(NTR), which displays a phenotypic down-regulation within CBF perikarya in AD. As pS422+ neurons increased in number, p75(NTR)+ neurons decreased, and these changes correlated with both AD neuropathology and cognitive decline. Also, NFTs developed slower in the CBF compared with previously examined cortical regions. Taken together, these results suggest that changes in cognition are associated with pretangle events within NB cholinergic neurons before frank NFT deposition.     

Adenosine inhibits glutamatergic input to basal forebrain cholinergic neurons

Adenosine has been proposed as an endogenous homeostatic sleep factor that accumulates during waking and inhibits wake-active neurons to promote sleep. It has been specifically hypothesized that adenosine decreases wakefulness and promotes sleep recovery by directly inhibiting wake-active neurons of the basal forebrain (BF), particularly BF cholinergic neurons. We previously showed that adenosine directly inhibits BF cholinergic neurons. Here, we investigated 1) how adenosine modulates glutamatergic input to BF cholinergic neurons and 2) how adenosine uptake and adenosine metabolism are involved in regulating extracellular levels of adenosine. Our experiments were conducted using whole cell patch-clamp recordings in mouse brain slices. We found that in BF cholinergic neurons, adenosine reduced the amplitude of AMPA-mediated evoked glutamatergic excitatory postsynaptic currents (EPSCs) and decreased the frequency of spontaneous and miniature EPSCs through presynaptic A(1) receptors. Thus we have demonstrated that in addition to directly inhibiting BF cholinergic neurons, adenosine depresses excitatory inputs to these neurons. It is therefore possible that both direct and indirect inhibition may synergistically contribute to the sleep-promoting effects of adenosine in the BF. We also found that blocking the influx of adenosine through the equilibrative nucleoside transporters or inhibiting adenosine kinase and adenosine deaminase increased endogenous adenosine inhibitory tone, suggesting a possible mechanism through which adenosine extracellular levels in the basal forebrain are regulated.     

Evidence for nerve growth factor-ganglioside interaction in forebrain cholinergic neurons

Cholinergic neurons of the forebrain respond trophically to nerve growth factor (NGF) in some experimental circumstances. The cholinergic cell system of the nucleus basalis magnocellularis (NBM) which projects to the cortex shows signs of cellular degeneration following limited devascularizing cortical lesions, while no apparent damage is observed in the remaining ipsilateral cortex. These cholinergic cells possess receptors for NGF and the administration of this peptide into the cerebroventricular space prevents cell shrinkage and loss of activity of the biosynthetic enzyme for acetylcholine, choline acetyltransferase (ChAT). Analogous trophic responses can be elicited in this system with the application of the sialoganglioside GM1. In addition, GM1 can increase the effects of NGF on ChAT activity in lesioned neurons of the NBM-to-cortex model system described above. This cooperative interaction is observed even when ineffective doses of GM1 are administered. Furthermore, an interaction between these two putative neurotrophic substances has been noted over other cholinergic parameters such as cortical high affinity choline uptake (HACU). These studies confirm the idea that trophic factors can be utilized to rescue degenerating neurons of the CNS and, in addition, lend support to the concept that gangliosides can facilitate actions of endogenously produced trophic factors.     

Adenosine inhibits basal forebrain cholinergic and noncholinergic neurons in vitro

Adenosine has been proposed as a homeostatic "sleep factor" that promotes the transition from waking to sleep by affecting several sleep-wake regulatory systems. In the basal forebrain, adenosine accumulates during wakefulness and, when locally applied, suppresses neuronal activity and promotes sleep. However, the neuronal phenotype mediating these effects is unknown. We used whole-cell patch-clamp recordings in in vitro rat brain slices to investigate the effect of adenosine on identified cholinergic and noncholinergic neurons of the magnocellular preoptic nucleus and substantia innominata. Adenosine (0.5-100 microM) reduced the magnocellular preoptic nucleus and substantia innominata cholinergic neuronal firing rate by activating an inwardly rectifying potassium current that reversed at -82 mV and was blocked by barium (100 microM). Application of the A1 receptor antagonist 8-cyclo-pentyl-theophylline (200 nM) blocked the effects of adenosine. Adenosine was also tested on two groups of electrophysiologically distinct noncholinergic magnocellular preoptic nucleus and substantia innominata neurons. In the first group adenosine, via activation of postsynaptic A1 receptors, reduced spontaneous firing via inhibition of the hyperpolarization-activated cation current. Blocking the H-current with ZD7288 (20 microM) abolished adenosine effects on these neurons. The second group was not affected by adenosine. These results demonstrate that, in the magnocellular preoptic nucleus and substantia innominata region of the basal forebrain, adenosine inhibits both cholinergic neurons and a subset of noncholinergic neurons. Both of these effects occur via postsynaptic A1 receptors, but are mediated downstream by two separate mechanisms.     

植物雌激素对去卵巢大鼠基底前脑胆碱能神经元的影响

目的：观察植物雌激素对去卵巢大鼠基底前脑胆碱能神经元表达的影响，探讨植物雌激素在中枢神经系统的保护作用及机制。方法：采用乙酰胆碱转移酶(ChAT)免疫组织化学ABC法，观察去卵巢大鼠5w后各组基底前脑内侧隔核(MS)，斜角带垂直支(VDB)胆碱能神经元的数目。结果：与去卵巢对照组相比，植物雌激素用药组、雌激素用药组的内侧隔核，斜角带垂直支胆碱能神经元数目明显升高(P＜0．05)，与假手术组差别不明显。结论：本研究提示植物雌激素能明显增加去卵巢大鼠基底前脑胆碱能神经元的表达，从而对中枢神经系统退行性病变起保护作用，并有望预防和治疗老年性痴呆。     

A decrease in size and number of basal forebrain cholinergic neurons is paralleled by diminished hippocampal cholinergic innervation in mice lacking leukocyte common antigen-related protein tyrosine phosphatase activity

The leukocyte common antigen-related (LAR) receptor, composed of an extracellular region with three immunoglobulin-like and eight fibronectin type III-like domains, and a cytoplasmic region containing two protein tyrosine phosphatase domains, is thought to play a role in axonal outgrowth and guidance during neural development. LAR mutant mice were generated completely lacking the two cytoplasmic protein tyrosine phosphatase domains, resulting in the loss of ability to bind intracellular associating proteins, but (may be) still containing the ability to perform extracellular functions. A reduction in size of basal forebrain cholinergic neurons and diminished hippocampal innervation reported for knockout mice that contain a leaky gene trap inserted into the 5' part of the LAR gene [Yeo T. T. et al. (1997) J. Neurosci. Res. 47, 348-360] warranted a computer-assisted quantitative image analysis throughout the basal forebrain and hippocampus of our LAR mutant mice. The total number, longest diameter and cell body area were calculated for the choline acetyltransferase-positive neurons in the medial septum and vertical diagonal band, and optical density measurements were performed to determine the extent of acetyl cholinesterase-positive fibre innervation of the different layers in the dentate gyrus. In LAR mutant mice, the number of cholinergic cells was significantly reduced (approximately 25%) in the vertical diagonal band. Also, the cross-sectional area of the cholinergic neurons in the medial septum and vertical diagonal band was reduced (5%). These findings were paralleled by a diminished cholinergic innervation of the supragranular (18%) and molecular (4%) layers of the dentate gyrus. Thus, LAR protein tyrosine phosphatase activity appears crucial for size, number and target projection of basal forebrain cholinergic neurons, further strengthening a role for LAR in CNS development.     

Cellular mechanisms for amyloid beta-protein activation of rat cholinergic basal forebrain neurons

The deposition of amyloid beta-protein (Abeta) in the brain and the loss of cholinergic neurons in the basal forebrain are two pathological hallmarks of Alzheimer's disease (AD). Although the mechanism of Abeta neurotoxicity is unknown, these cholinergic neurons display a selective vulnerability when exposed to this peptide. In this study, application of Abeta(25-35) or Abeta(1-40) to acutely dissociated rat neurons from the basal forebrain nucleus diagonal band of Broca (DBB), caused a decrease in whole cell voltage-activated currents in a majority of cells. This reduction in whole cell currents occurs through a modulation of a suite of potassium conductances including calcium-activated potassium (I(C)), the delayed rectifier (I(K)), and transient outward potassium (I(A)) conductances, but not calcium or sodium currents. Under current-clamp conditions, Abeta evoked an increase in excitability and a loss of accommodation in cholinergic DBB neurons. Using single-cell RT-PCR technique, we determined that Abeta actions were specific to cholinergic, but not GABAergic DBB neurons. Abeta effects on whole cell currents were occluded in the presence of membrane-permeable protein tyrosine kinase inhibitors, genistein and tyrphostin B-44. Our data indicate that the Abeta actions on specific potassium conductances are modulated through a protein tyrosine kinase pathway and that these effects are selective to cholinergic but not GABAergic cells. These observations provide a cellular basis for the selectivity of Abeta neurotoxicity toward cholinergic basal forebrain neurons that are at the epicenter of AD pathology.     

Basal forebrain cholinergic cell attachment and neurite outgrowth on organotypic slice cultures of hippocampal formation

Distributions of somata and neurites of cholinergic neurons were studied after seeding dissociated cells onto organotypic slice cultures. Slice cultures were made from hippocampal formation and adjacent cortical regions from rats or mice. Dissociated cell suspensions of basal forebrain tissue from rat or mouse fetuses were seeded onto the slice cultures. Combined cultures were maintained for 1-21 days in vitro. Cultures processed for acetylcholinesterase (AChE) histochemistry demonstrated non-random patterns of cholinergic cells and their neurites. Labeled cells appeared most frequently in the molecular layer of the dentate gyrus, and in the deeper layers of cortical regions adjacent to the hippocampus. Neurites extending from these labeled cells appeared to target the dentate molecular layer and the cortical subplate layer. By 4 days in vitro, AChE-positive basal forebrain cells display several short and thick neurites that appear to be dendrites, and one long process that appears to be an axon. By 5 days in vitro, dendrites are well developed; by 7 days the presumed axon has extended widely over the cortical target zone. These neurites are maintained through 3 weeks in culture. Distributions of cells varied with the age of the slice. AChE-labeled cells were not seen overlying hippocampal tissue when dissociated cells were seeded on slice cultures made from day 0 rats, but a few labeled cells were seen when seeded on slices from day 2 rats. Clear non-random patterns of labeled cells and neurite outgrowth were seen on slice cultures from day 5 or older pups. The non-random distribution seen with AChE-positive neurons was not seen using other techniques that labeled all cells (non-selective fluorescent labels) or all neurons; these techniques resulted in labeled cells scattered apparently homogenously across the slice culture.These studies demonstrate a non-random pattern of attachment or differentiation of basal forebrain cholinergic neurons when these cells are seeded onto cultured cortical slices; this pattern mimics the normal patterns of basal forebrain cholinergic projections to these cortical regions. These data suggest that the factors that normally guide basal forebrain-derived cholinergic axons to their target cells in vivo are present and detectable in this model system.     

Development of the basal forebrain cholinergic system: phenotype expression prior to target innervation.

We measured choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in the rat to determine the time course of development, maturity, and senescence of ChAT activity. Tissue was obtained from Sprague-Dawley rats ranging in age from embryonic day 14 through 23 months. Seven regions were examined, including the magnocellular preoptic/substantia innominata region, frontal cortex, medial septal region, hippocampus, diagnoal band, and medial and lateral striatum. ChAT and AChE activities were first detected as early as E18 in the medial septum, diagonal band and magnocellular preoptic area, all regions of cholinergic cell bodies. Enzyme activity subsequently developed in terminal fields of these cholinergic perikarya (hippocampus and frontal cortex) as well as in the striatum. For all regions, enzyme activity rose during the first four postnatal weeks. This increase in enzyme activity was transient and, in most instances, decreases were observed between postnatal days 30 and 60. Most dramatic were the decreases in enzyme activity in the magnocellular preoptic/substantia innominata and diagonal band regions. Age-related declines also occurred in the frontal cortex, hippocampus, magnocellular preoptic/substantia innominata region, and the striatum. Cholinergic systems undergo dynamic changes especially during development and adulthood.     

Genistein对去卵巢大鼠基底前脑胆碱能神经元影响的体内研究

本文旨在研究染料木素(genistein)对去卵巢大鼠基底前脑胆碱能神经元的影响。雌性大鼠双侧卵巢切除2周后用genistein和雌激素替代治疗1周。称子宫重量以确定手术是否成功及雌二醇(E2)的治疗是否有效。用免疫组化染色、RT-PCR和Westernblot等方法对胆碱能神经元数量、ChAT基因和蛋白的表达量进行检测。结果显示:去卵巢3周后子宫变轻,雌激素替代治疗能增加去卵巢子宫的重量,而genistein替代治疗对去卵巢子宫的重量影响不明显;去卵巢3周后,内侧隔核(MS)和斜角带垂直臂核(VDB)内的胆碱能神经元数量、ChAT基因和蛋白的表达量均明显减少,雌激素和genistein替代治疗后能显著增加去卵巢大鼠MS和VDB内的胆碱能神经元数量、ChAT基因和蛋白的表达量。本研究结果提示:genistein对去卵巢大鼠基底前脑胆碱能神经元具有类似雌激素样神经保护作用,而对子宫影响不明显。     

Estrogen-mediated regulation of cholinergic expression in basal forebrain neurons requires extracellular-signal-regulated kinase activity

Beyond the role estrogen plays in neuroendocrine feedback regulation involving hypothalamic neurons, other roles for estrogen in maintaining the function of CNS neurons remains poorly understood. Primary cultures of embryonic rat neurons together with radiometric assays were used to demonstrate how estrogen alters the cholinergic phenotype in basal forebrain by differentially regulating sodium-coupled high-affinity choline uptake and choline acetyltransferase activity. High-affinity choline uptake was significantly increased 37% in basal forebrain cholinergic neurons grown in the presence of a physiological dose of estrogen (5 nM) from 4 to 10 days in vitro whereas choline acetyltransferase activity was not significantly changed in the presence of 5 or 50 nM estrogen from 4 to 10 or 10 to 16 days in vitro. Newly-synthesized acetylcholine was significantly increased 35% following 6 days of estrogen treatment (10 days in vitro). These effects are in direct contrast to those found for nerve growth factor; that is, nerve growth factor can enhance the cholinergic phenotype through changes in choline acetyltransferase activity alone. This is most surprising given that mitogen-activated protein kinase and extracellular-signal-regulated kinase1/2, kinases also activated in the signaling pathway of nerve growth factor, were found to participate in the estrogen-mediated changes in the cholinergic phenotype. Likewise, general improvement in the viability of the cultures treated with estrogen does not account for the effects of estrogen as determined by lactate dehydrogenase release and nerve growth factor-responsiveness. These findings provide evidence that estrogen enhances the differentiated phenotype in basal forebrain cholinergic neurons through second messenger signaling in a manner distinct from nerve growth factor and independent of improved survival.     

秋水仙碱对基底前脑巢蛋白阳性和阴性胆碱能神经元的影响

目的:通过研究侧脑室注射秋水仙碱前后基底前脑巢蛋白阳性和阴性胆碱能神经元变化,探讨巢蛋白表达对基底前脑神经元机能的影响及其可能机制。方法:成年健康雌性SD大鼠随机分为正常对照组和侧脑室注射秋水仙碱组,术后分别于24 h、48 h、3 d、7 d、14 d和28 d取脑行冷冻切片与免疫组织化学显色,比较秋水仙碱注射后不同时间点基底前脑巢蛋白~+和巢蛋白~-胆碱能神经元的数目变化。结果:大鼠侧脑室秋水仙碱注射后24 h,基底前脑的内侧隔核(MS)、斜角带核垂直支(vDB)和水平支(hDB)的巢蛋白~+和巢蛋白的胆碱能神经元数目都急骤下降。随着时间的推移巢蛋白~+神经元数目逐渐恢复,术后14 d,巢蛋白~+神经元数目基本恢复至正常水平,但巢蛋白~-胆碱能神经元数目一直维持较低水平。结论:侧脑室注射秋水仙碱后,基底前脑巢蛋白~+和巢蛋白~-的胆碱能神经元都急骤减少,但巢蛋白~+神经元在减少后可逐渐恢复,而巢蛋白~-神经元则不能恢复。     

雌激素对去卵巢大鼠基底前脑胆碱能神经元及一氧化氮合酶阳性神经元的影响

目的　探讨雌激素补充治疗对去卵巢大鼠胆碱能神经元及一氧化氮合酶 (NOS)阳性神经元表达的影响及剂量效应关系。方法　 2 0只去卵巢SD大鼠分成 4个不同剂量组 :0 μg(对照组 )、2 0 μg(0 0 8mg/kg)、5 0 μg(0 2 0mg/kg)、10 0 μg(0 4 0mg/kg)组 ;1周后 ,用乙酰胆碱转移酶 (ChAT)免疫化学方法及尼克酰胺腺嘌呤二核苷酸黄递酶 (NADPH d)组织化学方法研究。结果　NOS阳性神经元在内侧隔核 (MS) ,其数目在各组间无明显差异 (P >0 0 5 )。在斜角带垂直支 (VDB) ,5 0 μg剂量组与对照组相比有明显差异 (P <0 0 5 ) ;ChAT阳性神经元在内侧隔核 (MS) ,其 2 0 μg、5 0 μg剂量组数目出现剂量递增效应 ,与对照组比较有明显差异 (P <0 0 5 ) ,但 10 0 μg剂量组与对照组比较没有明显差异 (P >0 0 5 ) ,同时 5 0 μg与 10 0 μg剂量组比较有明显差异 (P <0 0 5 )。ChAT阳性神经元在斜角带水平支 (HDB) ,各组间均无明显差异 (P >0 0 5 )。结论　雌激素补充治疗能选择性影响基底前脑各亚区NOS和胆碱能神经元 ,并有可能影响学习和记忆能力。     

A functional role of cholinergic innervation to neurons in the cat visual cortex

1. Effects of microionophoretic application of acetylcholine (ACh) and its antagonists on neuronal responses to visual stimuli and to electrical stimulation of the lateral geniculate nucleus were studied in the cat striate cortex. 2. Responses elicited visually and electrically were facilitated by ACh in 74% of the cells tested, whereas the responses were suppressed in 16%. These ACh effects were blocked by a muscarinic antagonist, atropine, but not by a nicotinic antagonist, hexamethonium, indicating that the ACh effects are mediated through muscarinic receptors. A single application of atropine suppressed visual responses of cells facilitated by ACh, whereas it enhanced those of cells inhibited by ACh, suggesting that endogenous ACh may tonically modulate visual responsivity of cortical neurons. 3. In most cells with the facilitatory ACh effect, responses with single spikes to the electrical stimulation became more consistent, often with double spikes, during the ACh application. The suppressive effects of ACh were noted most often in cells with a longer response latency to electrical stimulation of lateral geniculate nucleus. 4. In most of the facilitated cells the spontaneous activity remained null or very low during ACh application, in spite of marked enhancement of visual responses, suggesting that ACh may improve the signal-to-noise ratio (S/N) of cortical neuron activity. To confirm this suggestion, we calculated a S/S + N index by counting the total number of spikes in the responses (S) and that in peristimulus time histogram (S + N) and found that it was improved during the ACh application in about a half of the cells, whereas it became worse in about one-fifth. 5. In most of the facilitated cells, ACh enhanced visual responses not only to optimal but also to nonoptimal stimuli, resulting in no improvement or even worsening of the orientation selectivity. This was also the case in the selectivity of direction of stimulus movement. 6. The laminar location of the facilitated cells was biased toward layers V and VI of the cortex, although they also made up the majority in layers II + III and about half the tested cells in layers IVab and IVc. 7. In the light of recent understanding of cortical circuitry, these results suggest that the cholinergic innervation to cortical neurons may play a role in improvement of the S/N ratio of information processing in the striate cortex and in facilitation of sending processed informations to other visual centers.     

Dissociation between the attentional functions mediated via basal forebrain cholinergic and GABAergic neurons

The role of basal forebrain corticopetal cholinergic projections in attentional functions has been extensively investigated. For example, 192 IgG-saporin-induced loss of cortical cholinergic inputs was repeatedly demonstrated to result in a selective impairment in the ability of rats to detect signals in a task designed to assess sustained attention performance. The loss of cortical cholinergic inputs correlated highly with the decrease in the hit rate. Little is known about the functions of basal forebrain non-cholinergic neurons, particularly corticopetal GABAergic neurons, largely because of the absence of specific research tools to manipulate selectively this projection. As basal forebrain lesions produced with ibotenic acid were previously observed to potently destroy non-cholinergic, particularly GABAergic neurons while producing only moderate decreases in the density of cortical cholinergic inputs, the present experiment examined the effects of such lesions on sustained attention performance and then compared these effects with the immunohistochemical and attentional consequences of selective cholinotoxic lesions produced by intra-basal forebrain infusions of 192 IgG-saporin. In contrast to the selective decrease in hits previously observed in 192 IgG-saporin-lesioned animals, the attentional performance of ibotenic acid-lesioned animals was characterized by a selective increase in the relative number of false alarms, that is 'claims' for signals in non-signal trials. Analyses of the response latencies suggested that this effect of ibotenic acid was due to impairments in the animals' ability to switch from the processing of the response rules for signal trials to those for non-signal trials. As expected, 192 IgG-saporin did not affect the number of basal forebrain parvalbumin-positive neurons, that are presumably GABAergic, but decreased cortical acetylcholinesterase-positive fiber density by over 80%. Conversely, in ibotenic acid-lesioned animals, basal forebrain parvalbumin-positive cells were decreased by 60% but cortical acetylcholinesterase-positive fiber density was only moderately reduced (less than 25%).These data form the basis for the development of the hypothesis that basal forebrain GABAergic neurons mediate executive aspects of attentional task performance. Such a function may be mediated in parallel via basal forebrain GABAergic projections to the cortex and the subthalamic nucleus.     

Stability of numbers but not size of mouse forebrain cholinergic neurons to 53 months

In normal mammalian aging there is a reduction of cholinergic markers in a variety of regions. To determine whether this reduction is related to reduced numbers of basal forebrain cholinergic neurons, we counted the number and measured the sizes of the magnocellular acetylcholinesterase-positive neurons in this region of 7, 15, and 53-month-old C57B1/6NNIA mice. Data were collected from coded slides containing the medial septum, nucleus of the diagonal band, magnocellular preoptic nucleus, and nucleus basalis magnocellularis. There was no decline in numbers of basal forebrain acetylcholinesterasepositive neurons in any of the regions studied. However, cell sizes showed a progressive age-related decline which was greatest in the nucleus basalis magnocellularis.     

Neonatal lesion of forebrain cholinergic neurons: further characterization of behavioral effects and permanency

Intraventricular injections of 192 IgG-saporin in the neonatal rat caused severe loss of basal forebrain cholinergic neurons and ectopic hippocampal ingrowths. These were evident at 24 months of age and thus, were lifelong consequences of the 192 IgG-saporin treatment. When tested as young adults on a novel water-escape radial arm maze, the rats with this lesion were slower to learn the task, committing significantly more working and reference memory errors before they achieved control level of performance. It is unlikely that this was a result of attentional impairment as the lesioned rats performed as vigilantly as controls in a five choice serial reaction time task. When tested in the Morris water maze at 22 months of age, they were slower at learning the hidden platform location. This contrasts with previous studies which have repeatedly shown that they normally acquire this task as young adults. It was concluded that this neonatal cholinergic lesion has modest but discernable effects on problem solving in young adulthood that are consistent with the reported effects of the lesion on cortical pyramidal neurons. The cognitive effects of the lesion may become more severe in aging, perhaps as a result of the added effects of aging on these neurons.