Nicotine increases the expression of neurotrophin receptor tyrosine kinase receptor A in basal forebrain cholinergic neurons

In this study, we investigated whether the potential positive effects of nicotine in Alzheimer's disease (AD) may involve neurotrophic factors, such as nerve growth factor (NGF), closely associated with basal forebrain (BF) cholinergic function and survival. To this aim, we studied the effects of prolonged nicotine treatment on neurotrophin receptors expression and on NGF protein levels in the rat BF cholinergic circuitry. Both in vivo and in vitro experiments were conducted. We found that s.c. nicotine infusion (1.2 mg free base/kg/d delivered by mini-pumps for 7 days) induced in vivo an increase in tyrosine kinase receptor A (TrkA)—but not TrkB, TrkC or low affinity neurotrophin receptor p75 (p75)—expression in BF cholinergic neurons targeting the cerebral cortex. Nicotine did not produce statistically significant long-lasting effects on NGF levels in the cerebral cortex, or in the BF. In vitro experiments performed on primary BF neuronal cultures, showed that 72 h exposure to nicotine increased both TrkA expression, and NGF release in culture medium. Neutralization experiments with an anti-NGF antibody showed that NGF presence was not necessary for nicotine-induced increase of TrkA levels in cultured cholinergic neurons, suggesting that nicotine may act through NGF-independent mechanisms. This study shows that nicotine, independently of its action on NGF levels, may contribute to the restoration of the trophic support to BF cholinergic neurons by increasing TrkA levels.     

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.     

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。     

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。     

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.     

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。     

Activation of basal forebrain cholinergic neurons differentially regulates brain-derived neurotrophic factor mRNA expression in different projection areas.

Afferent cholinergic pathways from the basal forebrain were activated by injections of the glutamate analog quisqualate either into the nucleus basalis or into the medial septal nucleus. Nucleus basalis injections had no effect on the expression of brain-derived neurotrophic factor (BDNF) mRNA in its neocortical projection areas as measured by in situ hybridization. In contrast, 7 h after an injection into the septum the level of BDNF mRNA increased 3- to 5-fold in the dentate gyrus, throughout CA1 to CA3 in the hippocampus and in the piriform cortex.     

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.     

The basal forebrain cholinergic system in the raccoon

The distribution of neurons displaying choline acetyltransferase (ChAT) immunoreactivity was examined in the raccoon basal forebrain using a rabbit antiscrum and a monoclonal antibody. Alternating sections were used for Nissl staining. ChAT-positive neurons were arranged in a continuous mass extending from the medial septum to the caudal pole of the pallidum. Based upon spatial relations to fibre tracts, the clustering of neuronal groups, and cytological criteria, the basal forebrain magnocellular complex can be subdivided into several distinct regions. Although clear nuclear boundaries were often absent, the ChAT-positive neurons were divided into: the nucleus tractus diagonalis (comprising pars septi medialis, pars verticalis and pars horizontalis); nucleus praeopticus magnocellularis; substantia innominata; and the nucleus basalis of Meynert. Comparison with Nissl-stained sections indicated the presence of varying proportions of non-cholinergic neurons clustered or arranged loosely within these basal forebrain subdivisions. These data provide a structural basis for studies concerned with the topographical and physiological aspects of the raccoon basal forebrain cholinergic projections and its comparison with the basal forebrains of other species.     

Genistein对大鼠基底前脑胆碱能神经元发育影响的体外研究

本文旨在研究染料木素(genistein)对体外培养的基底前脑胆碱能神经元发育的影响。取孕18d胎鼠基底前脑神经元,体外有血清培养7d后,随机分为3组:无血清培养液组(control组),genistein+无血清培养液组(G组)、E2+无血清培养液组(E2组)。48h后用MTT法测定细胞活力和代谢状态;用AChE组化染色以及ChAT和MAP2免疫荧光双重染色,镜下计数AChE阳性和ChAT阳性神经元数,并对两种神经元的细胞面积、第一级突起数及最长突起长度进行检测。结果显示:MTT法所测的OD值、AChE阳性和ChAT阳性神经元数、细胞面积、第一级突起数及最长突起长度在G组与control组之间无明显差异,然而E2组中的上述数值均明显增加。本研究的结果提示:genistein对体外培养的基底前脑胆碱能神经元无类似雌激素样的神经保护和营养作用。     

Human amylin actions on rat cholinergic basal forebrain neurons: antagonism of beta-amyloid effects

Human amylin (hAmylin), a 37-amino acid pancreatic peptide, and amyloid beta protein (A beta), a 39-43 amino acid peptide, abundantly deposited in the brains of Alzheimer's patients, induce neurotoxicity in hippocampal and cortical cultures. Although the mechanism of this neurotoxicity is unknown, both peptides are capable of modulating ion channel function that may result in a disruption of cellular homeostasis. In this study, we examined the effects of hAmylin on whole cell currents in chemically identified neurons from the rat basal forebrain and the interactions of hAmylin-induced responses with those of A beta. Whole cell patch-clamp recordings were performed on enzymatically dissociated neurons of the diagonal band of Broca (DBB), a cholinergic basal forebrain nucleus. Bath application of hAmylin (1 nM to 5 microM) resulted in a dose-dependent reduction in whole cell currents in a voltage range between -30 and +30 mV. Single-cell RT-PCR analysis reveal that all DBB neurons responding to hAmylin or A beta were cholinergic. Using specific ion channel blockers, we identified hAmylin and A beta effects on whole cell currents to be mediated, in part, by calcium-dependent conductances. Human amylin also depressed the transient outward (IA) and the delayed rectifier (IK) potassium currents. The hAmylin effects on whole cell currents could be occluded by A beta and vice versa. Human amylin and A beta responses could be blocked with AC187 (50 nM to 1 microM), a specific antagonist for the amylin receptor. The present study indicates that hAmylin, like A beta, is capable of modulating ion channel function in cholinergic basal forebrain neurons. Furthermore, the two peptides may share a common mechanism of action. The ability of an amylin antagonist to block the responses evoked by hAmylin and A beta may provide a novel therapeutic approach for Alzheimer's disease.     

Morphological characterization of electrophysiologically and immunohistochemically identified basal forebrain cholinergic and neuropeptide Y-containing neurons

The basal forebrain (BF) contains cholinergic as well as different types of non-cholinergic corticopetal neurons and interneurons, including neuropeptide Y (NPY) containing cells. BF corticopetal neurons constitute an extrathalamic route to the cortex and their activity is associated with an increase in cortical release of the neurotransmitter acetylcholine, concomitant with low voltage fast cortical EEG activity. It has been shown in previous studies (Duque et al. in J Neurophysiol 84:1627–1635, 2000) that in anesthetized rats BF cholinergic neurons fire mostly during low voltage fast cortical EEG epochs, while increased NPY neuronal firing is accompanied by cortical slow waves. In this paper, electrophysiologically and neurochemically characterized cholinergic and NPY-containing neurons were 3D reconstructed from serial sections and morphometrically analyzed. Cholinergic and NPY-containing neurons, although having roughly the same dendritic surface areas and lengths, were found to differ in dendritic thickness and branching structure. They also have distinct patterns of dendritic endings. The subtle differences in dendritic arborization pattern may have an impact on how synaptic integration takes place in these functionally distinct neuronal populations. Cholinergic neurons exhibited cortically projecting axons and extensive local axon collaterals. Elaborate local axonal arbors confined to the BF also originated from NPY-containing neurons. The presence of local axon collaterals in both cholinergic and NPY neurons indicates that the BF is not a mere conduit for various brainstem inputs to the cortex, but a site where substantial local processing must take place. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Age-related sex differences in spatial learning and basal forebrain cholinergic neurons in F344 rats

Basal forebrain cholinergic neurons are important for spatial learning in rodents. Spatial learning ability is reportedly better in males than females, and declines with age. To examine the role of cholinergic function in sex- or age-related differences in spatial learning, we compared the size of basal forebrain cholinergic neurons (BFCN) of young and aged male and female Fischer 344 (F344) rats that had been trained in the Morris water maze. Young male and female rats were equally proficient in finding the platform during training trials, but probe tests revealed that young male rats had better knowledge of the platform's precise location. Impairments in spatial learning were observed in aged rats, and the advantage of males over females was lost. BFCN were significantly larger in young male than young female rats, and were correlated with spatial memory performance for both groups. BFCN were smaller in aged than young males; no change was seen between young and aged females. In the groups of aged rats the correlation between neuron size and spatial memory was lost. The present findings provide further evidence of a role for the basal forebrain cholinergic system in spatial learning, but reveal a complex interaction between sex, age and behavioral performance.     

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。     

Non-cholinergic basal forebrain neurons project to the contralateral basal forebrain in the rat

Following injections of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) or the fluorescent tracer fluoro-gold into the magnocellular preoptic area and the horizontal limb of the diagonal band, retrogradely labelled neurons were found in the homotopic region of the contralateral basal forebrain. Labelled fibers apparently arising from these neurons travelled in the stria medullaris and the habenular commissure to terminate in the contralateral basal forebrain. Although the neurons retrogradely labelled with fluoro-gold in the contralateral basal forebrain were similar in size to choline acetyltransferase (ChAT)-immunoreactive neurons, and were intermingled with them, none was ChAT-positive. WGA-HRP injections into the nucleus basalis magnocellularis did not result in retrograde labelling in the contralateral basal forebrain. These findings suggest that non-cholinergic neurons may serve as a direct link between the two sides of selective magnocellular basal forebrain regions.     

Somatostatin presynaptically inhibits both GABA and glutamate release onto rat basal forebrain cholinergic neurons

A whole cell patch-clamp study was carried out in slices obtained from young rat brain to elucidate the roles of somatostatin in the modulation of synaptic transmission onto cholinergic neurons in the basal forebrain (BF), a region that contains cholinergic and GABAergic corticopetal neurons and somatostatin (SS)-containing local circuit neurons. Cholinergic neurons within the BF were identified by in vivo prelabeling with Cy3 IgG. Because in many cases SS is contained in GABAergic neurons in the CNS, we investigated whether exogenously applied SS can influence GABAergic transmission onto cholinergic neurons. Bath application of somatostatin (1 muM) reduced the amplitude of the evoked GABAergic inhibitory presynaptic currents (IPSCs) in cholinergic neurons. SS also reduced the frequency of miniature IPSCs (mIPSCs) without affecting their amplitude distribution. SS-induced effect on the mIPSC frequency was significantly larger in the solution containing 7.2 mM Ca(2+) than in the standard (2.4 mM Ca(2+)) external solution. Similar effects were observed in the case of non-NMDA glutamatergic excitatory postsynaptic currents (EPSCs). SS inhibited the amplitude of evoked EPSCs and reduced the frequency of miniature EPSCs dependent on the external Ca(2+) concentration with no effect on their amplitude distribution. Pharmacological analyses using SS-receptor subtype-specific drugs suggest that SS-induced action of the IPSCs is mediated mostly by the sst(2) subtype, whereas sst subtypes mediating SS-induced inhibition of EPSCs are mainly sst(1) or sst(4). These findings suggest that SS presynaptically inhibits both GABA and glutamate release onto BF cholinergic neurons in a Ca(2+)-dependent way, and that SS-induced effect on IPSCs and EPSCs are mediated by different sst subtypes.     

Age-related recurrence of basal forebrain lesion-induced cholinergic deficits

Lesions of basal forebrain cholinergic nuclei projecting in neocortex have recently been employed as an animal model for the cholinergic deficits in Alzheimer's disease. However, unlike Alzheimer's patients, whose deterioration appears to be progressive and irreversible, basalis lesioned rats usually recover both behaviorally and neurochemically within several months after the lesion. We now demonstrate that this recovery may be a function of the age of the rat and that cholinergic deficits re-occur in the aged rat. Choline acetyltransferase (ChAT) activity and [3H]hemicholinium-3 ([3H]HCh-3) binding are reduced in cortex ipsilateral to ibotenic acid lesions in the 12-month postlesion rat following an initial recovery to normal levels by about 3 months postlesion. The recurrence of decrease of cholinergic markers is not a consequence of a non-specific age-related decline since the activity of glutamic acid decarboxylase remains constant between 3 and 12 months postlesion.     

Model for aging in the basal forebrain cholinergic system

A key component of the cognitive deficits associated with aging is the loss of function of cholinergic neurons in the basal forebrain due to neuronal losses and decreased cholinergic function of spared neurons. A model to mimic one aspect of this phenomenon is to kill cholinergic neurons selectively in the basal forebrain via administration of the immunotoxin IgG-192-saporin. Here we discuss apoptotic regulators, such as nerve growth factor, in age-associated changes present in the cholinergic system and the role of the NF-kappaB signaling system in cellular commitment to apoptosis. We also examine the age-associated decline in intrinsic response mechanisms, which may account for the age-associated reduction in recovery from both acute and chronic insults to the central nervous system.