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.     

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。     

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。     

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。     

七氟醚处理对老龄小鼠基底前脑胆碱能系统和认知功能的影响

目的:探讨七氟醚对基底前脑胆碱能系统和认知功能的影响。方法:老年C57小鼠吸入七氟醚建立全身麻醉动物模型,利用Morris水迷宫检测认知功能,免疫荧光检测Meynert基底核中胆碱能神经元计数,Western Blot检测额叶皮层胆碱乙酰转移酶及高亲和力胆碱转运体的蛋白水平,ELISA测定额叶皮层胆碱乙酰转移酶的活性。结果:与对照组相比,七氟醚组小鼠平台潜伏期延长,平台穿越次数减少(2.1±0.4 vs 5.6±0.5,P0.05),原平台所在象限停留时间缩短[(21.5±2.4)%vs(48.6±2.8)%,P0.05]。此外,七氟醚组小鼠Meynert基底核胆碱能神经元计数减少(3748±248 vs 5942±315,P0.05),额叶皮层胆碱乙酰转移酶及高亲和力胆碱转运体表达下降,伴有胆碱乙酰转移酶活性降低[(31.4±1.1)μmol/(g.h)vs(55.8±1.3)μmol/(g.h),P0.05]。结论:七氟醚可损害老龄鼠基底前脑胆碱能系统并诱导认知功能损害。     

胰岛素与尼莫地平对大鼠基底前脑胆碱能神经元及空间学习记忆的影响

目的探讨胰岛素与尼莫地平对基底前脑胆碱能神经元与学习记忆在1型糖尿病脑病发生中的影响。方法将成年雄性Wistar大鼠随机分成糖尿病组、干预组、载体组及正常对照组。用链脲佐菌素成功建立成1型糖尿病模型12周后,对干预组大鼠每日皮下注射长效胰岛素(3 IU)、腹腔注射尼莫地平(20 mg/kg),连续用药6周,在相同条件下对载体组大鼠注射等体积的无药物液体,但对糖尿病组或正常对照组大鼠未进行任何处理。应用Morris水迷宫及胆碱乙酰转移酶(ChAT)免疫组化方法,分别测定各组大鼠的空间学习记忆能力及基底前脑胆碱能神经元的变化。结果糖尿病组大鼠内侧隔核、斜角带核垂直支、斜角带核水平支的ChAT阳性神经元数均明显减少(P<0.05),空间学习记忆能力也明显下降(P<0.05);干预组大鼠以上三个核团的ChAT阳性神经元数与学习记忆能力均明显大于糖尿病组大鼠(P<0.05),但干预组的各项指标仍然明显低于载体组大鼠或正常对照组大鼠(P<0.05)。结论在糖尿病的长期自然发展过程中,若不进行治疗则可累及到基底前脑胆碱能神经元,导致学习记忆障碍,这可能是引发1型糖尿病脑病的一个负性因素;此时联合应用胰岛素与尼莫地平仍可有效遏制该脑病向纵深发展的恶性势头。     

Evidence for a distinct group of nestin-immunoreactive neurons within the basal forebrain of adult rats

Nestin is an intermediate filament protein serving as a marker for neuroprogenitor and stem cells. Here we report that a cluster of previously unrecognized nestin immunoreactive (nestin-ir) neurons was located in the medial septum-diagonal band of Broca (MS-DBB) of the basal forebrain in adult rats. Nestin-ir neurons were exclusively located in the MS-DBB and intermingled with choline acetyltransferase-ir (ChAT-ir), parvalbumin-ir (PV-ir), or nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase reactive (NADPHd-reactive) neurons. However, there was no colocalization between nestin-ir and PV-ir in single neurons in MS-DBB; only about 35% of nestin-ir neurons were ChAT-ir, and 8%-12% of nestin-ir neurons were NADPHd-reactive. Morphologically, nestin-ir neurons showed a larger size of somata than that of ChAT-ir or PV-ir neurons and the distribution of nestin-ir neurons spread across the rostro-caudal extent of the MS-DBB. Moreover, retrograde tracing revealed that a significant portion of these nestin-ir neurons projected to the thalamus and hippocampus. These results, for the first time, provide strong evidence that there exists a cluster of previously unrecognized nestin-ir neurons in MS-DBB of the basal forebrain in adult rats and that these nestin-ir neurons are distinguishable from ChAT-ir, PV-ir, and NADPHd-reactive neurons.     

Pontine cholinergic mechanisms and their impact on respiratory regulation

Activation of pontomedullary cholinergic neurons may directly and indirectly cause depression of respiratory motoneuronal activity, activation of respiratory premotor neurons and acceleration of the respiratory rate during REM sleep, as well as activation of breathing during active wakefulness. These effects may be mediated by distinct subpopulations of cholinergic neurons. The relative inactivity of cholinergic neurons during slow-wave sleep also may contribute to the depressant effects of this state on breathing. Cholinergic muscarinic and nicotinic receptors are expressed in central respiratory neurons and motoneurons, thus allowing cholinergic neurons to act on the respiratory system directly. Additional effects of cholinergic activation are mediated indirectly by noradrenergic, serotonergic and other neurons of the reticular formation. Excitatory and suppressant respiratory effects with features of natural states of REM sleep or active wakefulness can be elicited in urethane-anesthetized rats by pontine microinjections of the cholinergic agonist, carbachol. Carbachol models help elucidate the neural basis of respiratory disorders associated with central cholinergic activation.     

Pontine GABAergic pathways: role and plasticity in the hypoxic ventilatory response

The hypoxic ventilatory response (HVR) was compared before and after uni- and bi-lateral injections of bicuculline, a GABA(A) receptor antagonist, into the ventrolateral (vl) pons and before and after conditioning animals to chronic sustained hypoxia (CSH). The HVR was assessed by recording phrenic nerve activity (PNA) during and after brief exposures to hypoxia (8% O(2) and 92% N(2) for 45s). Inspiratory (T(I)) and expiratory (T(E)) durations were averaged before hypoxia, at the peak breathing frequency during hypoxia, before the end of hypoxia, immediately after hypoxia, and 60s after hypoxia. Blocking GABA(A) receptors in the vl pons prolonged T(E) during, but not after hypoxia. After CSH induced by 14 days in a hypobaric chamber (0.5atm), the HVR was attenuated compared to that in the naive animals. This plasticity of HVR was associated with selective induction of alpha6 and delta GABA(A) receptor subunit mRNAs specifically in the pons compared to the medulla. These physiological and molecular results illustrate the importance of pontine GABAergic pathways in shaping the response to hypoxia.     

Cholinergic projections from the basal forebrain to the frontal cortex: a combined fluorescent tracer and immunohistochemical analysis in the rat

Cholinergic projections from the basal forebrain to some regions of the frontal cortex were studied by infusing propidium iodide (PI), a fluorescent tracer, into areas 6 and 10 and microscopically assessing the cellular co-localization of PI and immunohistochemically demonstrated choline-O-acetyltransferase (ChAT). The same brain sections were additionally processed for acetylcholinesterase (AChE, pharmacohistochemical regimen) and Nissl material (cresyl violet stain). Basal forebrain neurons projecting to the frontal cortex were found primarily in nucleus basalis, but others were located in association with the substantia innominata/lateral preoptic area, magnocellular preoptic area, and ansa lenticularis. These projection neurons were large (>25 μm in maximum soma extent), demonstrated ChAT-like immunoreactivity, stained intensely for AChE following systemic administration of bis-(1-methyl-ethyl)phosphorofluoridate, and were highly chromophilic.     

单侧磨牙缺失对老年学习记忆减退大鼠基底前脑胆碱能系统的影响

目的:探讨单侧缺牙对不同学习记忆能力老年大鼠基底前脑胆碱能系统的影响。方法:用Morris水迷宫筛选出老年记忆减退鼠和老年记忆正常鼠,拔除单侧磨牙后2个月,用免疫组化和组织化学染色观察对其斜角带核垂直支胆碱能神经元和海马CA1区、前额皮质胆碱能纤维密度的影响。结果:斜角带核垂直支ChAT阳性细胞数和海马CA1区、前额皮质AChE阳性纤维密度在老年记忆减退鼠中,拔牙组较对照组(未拔牙)明显下降;海马CA1区AChE阳性纤维密度在老年记忆正常鼠中,拔牙组较对照组明显下降。结论:单侧磨牙缺失可加速老年学习记忆减退鼠基底前脑胆碱能系统的损害。     

Tachykinins in the control of breathing by hypoxia: pre- and post-genomic era

This article highlights major findings from physiological and pharmacological studies conducted in the pre- and post-genomic era examining the roles of substance P (SP) and other tachykinins in the response of the carotid body to hypoxia, in the ventilatory response to hypoxia and in respiratory rhythm generation. In the post-genomic period, the hypoxic ventilatory responses of mice carrying targeted deletion of genes that affect synthesis or degradation or receptor interaction of SP have been examined by us and also by other investigators. A brief summary of the findings from these investigations will also be presented. The combined observations from the pre- and post-genomic era strongly support the involvement of SP and also other tachykinins in the control of respiration during hypoxia.     

The role of cholinergic basal forebrain neurons in adenosine-mediated homeostatic control of sleep: lessons from 192 IgG-saporin lesions

A topic of high current interest and controversy is the basis of the homeostatic sleep response, the increase in non-rapid-eye-movement (NREM) sleep and NREM-delta activity following sleep deprivation (SD). Adenosine, which accumulates in the cholinergic basal forebrain (BF) during SD, has been proposed as one of the important homeostatic sleep factors. It is suggested that sleep-inducing effects of adenosine are mediated by inhibiting the wake-active neurons of the BF, including cholinergic neurons. Here we examined the association between SD-induced adenosine release, the homeostatic sleep response and the survival of cholinergic neurons in the BF after injections of the immunotoxin 192 immunoglobulin G (IgG)-saporin (saporin) in rats. We correlated SD-induced adenosine level in the BF and the homeostatic sleep response with the cholinergic cell loss 2 weeks after local saporin injections into the BF, as well as 2 and 3 weeks after i.c.v. saporin injections. Two weeks after local saporin injection there was an 88% cholinergic cell loss, coupled with nearly complete abolition of the SD-induced adenosine increase in the BF, the homeostatic sleep response, and the sleep-inducing effects of BF adenosine infusion. Two weeks after i.c.v. saporin injection there was a 59% cholinergic cell loss, correlated with significant increase in SD-induced adenosine level in the BF and an intact sleep response. Three weeks after i.c.v. saporin injection there was an 87% cholinergic cell loss, nearly complete abolition of the SD-induced adenosine increase in the BF and the homeostatic response, implying that the time course of i.c.v. saporin lesions is a key variable in interpreting experimental results. Taken together, these results strongly suggest that cholinergic neurons in the BF are important for the SD-induced increase in adenosine as well as for its sleep-inducing effects and play a major, although not exclusive, role in sleep homeostasis.     

Pontine influences on respiratory control in ectothermic and heterothermic vertebrates

Respiratory rhythm generators appear both evolutionarily and developmentally as paired segmental rhythm generators in the reticular formation, associated with the motor nuclei of cranial nerves V, VII, IX, X, and XII. Those associated with the Vth and VIIth motor nuclei are "pontine" in origin and in fishes that employ a buccal suction/force pump for breathing the primary pair of respiratory rhythm generators are associated with the trigeminal nuclei. In amphibians, while the basic respiratory pump remains the same, the dominant site of respiratory rhythm generation has been assumed by the facial, glossopharyngeal and vagal motor nuclei. In reptiles, birds and mammals, in general there is a switch to an aspiration pump driven by thoraco-lumbar muscles innervated by spinal nerves. In these groups, the critical sites necessary for respiratory rhythmogenesis now sit near the ponto-medullary border, in the parafacial region (which may underlie expiratory-dominated, intercostal-abdominal breathing in non-mammalian tetrapods) and in a more caudal region, the preBotzinger complex (which may underlie inspiratory-dominated diaphragmatic breathing in mammals).     

Sex steroid hormones and the neural control of breathing

We review evidence that sex steroid hormones including estrogen, progesterone and testosterone are involved in the central neural control of breathing. Sex hormones may exert their effects on respiratory motoneurons via neuromodulators, in particular, the serotonergic system. Recent studies have shown that levels of serotonin (5HT) in the hypoglossal and phrenic nuclei are greater in female than in male rats. Serotonin-dependent plasticity in hypoglossal and phrenic motor output also differs in male and female rats. Changing levels of gonadal hormones throughout the estrus cycle coincide with changing levels of 5HT in respiratory motor nuclei, and gonadectomy in male rats results in a decrease in 5HT-dependent plasticity in respiratory motor output. We speculate that sex steroid hormones are critically involved in adaptations in the neural control of breathing throughout life, and that decreasing levels of these hormones with increasing age may have a negative influence on the respiratory control system in response to challenge.     

Periodic breathing and genetics

The episodic waxing and waning of ventilation is a fundamental event in sleep apnea syndromes. Post-hypoxic frequency decline (PHFD) and periodic breathing (PB) are evoked by brief hypoxic exposures in unanaesthetized and unrestrained inbred C57BL/6J mice, but not in A/J mice; and expression of PHFD differs not only among these mice strains but in among rat strains as well. These observations along with the current literature on genetic factors that operate on ventilatory behavior at rest and with chemosensory drive lead to the hypothesis that genetic factors infer some proportion of risk for the ventilatory instability observed in human sleep apnea syndromes.     

Neurochemical perspectives on the control of breathing during sleep

A specific depression of minute ventilation occurs during sleep in normal subjects. This sleep-related ventilatory depression is partially related to mechanical events and upper airway atonia but some data also indicate that it is likely to be centrally mediated. This paper reviews the anatomical and neurochemical connections between sleep/wake- and respiratory-related areas in an attempt to identify the potential implication of sleep-related neurochemicals (serotonin, catecholamines, GABA, acetylcholine) in the sleep-related hypoventilation. The review of available data suggests that the sleep-related ventilatory depression depends upon the enhanced GABAergic activity together with a loss of suprapontine influence depending on the cessation of activity of the reticular formation. During REM sleep, an additional inhibitory activity emerges from the pontine cholinergic neurons, which contributes to the breathing irregularities and the associated depression of minute ventilation and ventilatory response to chemical stimuli. This model may contribute to a better understanding of the neurochemical environment of respiratory neurons during sleep, which remains a question of importance regarding the numerous pathological states that are linked to specific perturbations of breathing control during sleep.     

Respiratory pattern modulation by the pedunculopontine tegmental nucleus

This study demonstrates respiratory modulation caused by stimulation of the pedunculopontine tegmental nucleus (PPT), a structure not classically included in the pontine respiratory neuronal network. The long-lasting increase in variability of respiratory parameters following glutamate microinjection into PPT in anesthetized, spontaneously breathing Sprague Dawley rats was more pronounced under ketamine than nembutal anesthesia. The induced respiratory perturbations were characterized by intermittent apneas and increased variability of expiratory (TE) and total (TT) breath durations in all animals. Although the baseline spontaneous breathing patterns (mean values of all respiratory parameters and their variabilities) were equivalent under ketamine and nembutal anesthesia, different anesthetic agents did affect respiratory responses to PPT stimulation by glutamate in terms of latency, duration, and structure. We conclude that glutamatergic stimulation of PPT has a significant impact on the brainstem respiratory pattern generator.     

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.