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The basal forebrain (BF) is known to participate in the control of motion, attention, learning and memory, and it also plays a key role in sleep-wake regulation. Although there is a strong heterogeneity among neurons in the BF, the main types are cholinergic, gamma-aminobutyric acid (GABAergic) and glutamatergic neurons. This review provided the research progress in the regulation of sleep-wakefulness behavior by the 3 neurons in the BF. The cholinergic neurons play roles in activation of cortex and promote phase transition between sleep and wakefulness. The cortical projecting GABAergic neurons, which accept the projections from the adjacent cholinergic and glutamatergic neurons, contribute to awakening and the maintenance of normal wakefulness. The GABAergic interneurons may promote sleepiness by inhibiting the wake-active neurons which excite the cortical neurons. The glutamatergic neurons regulate sleep and wakefulness by interacting with neighbor cholinergic and cortical projecting GABAergic neurons or through the direct projection to the cortex as well.
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Aim To investigate the effects of neurotro-phin-3 ( NT-3 ) gene overexpression on the differentia-tion into cholinergic neuron of neural stem cells ( NSCs) in vitro and its underlying mechanism. Meth-ods Brain-derived NSCs from newborn mice were iso-lated and cultured in vitro and determined by immuno-fluorescence. The NSCs were divided into three groups: NSCs, GFP-NSCs and NT-3-NSCs groups. The expression of NT-3 was detected by immunofluo-rescence and ELISA. Then, the ability of NSCs on dif-ferentiation into cholinergic neuron was detected by im-munofluorescence and RT-PCR, and the Acetylcholine Assay Kit was used for acetylcholine ( ACh) , and the expression of Hes1 , Mash1 and Ngn1 mRNA was de-termined by RT-PCR. Results The neurosphere dis-played Nestin and Sox 2-postive by immunofluores-cence, suggesting that the cultured cells were NSCs. The proportion of ChAT immunopositive cells was sig-nificantly higher in the NT-3-NSCs group than that in the other two groups ( P <0. 01 ) . Ach secretion in NT-3-NSCs was significantly elevated compared with the other two groups ( P <0. 01 ) . NSCs transfected with NT-3 increased the levels of Mash1 and Ngn1 mR-NA, and decreased the level of Hes1 mRNA ( P <0. 05 ) . Conclusion NT-3 can significantly promote the in vitro differentiation of NSCs into cholinergic neu-rons via probablly inhibiting Notch signaling pathway.
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Medial pontine reticular formation (mPRF),an important part of brainstem reticular formation, has drawn great attention from anesthesiologists due to its close relation with not only consciousness, but also analgesia, muscle relaxation and autonomic reflex. Progress in related research may provide evidence for understanding the mechanism of general anesthesia; meanwhile,it may serve as an effective anesthesia target site, which may contribute to improvement in anesthetic effect and reduction of complications.
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Objective To establish a Alzheimer dementia(AD) model in mice. Methods The C57BL/6 mice were lesioned with ibotenic acid in Nucleus basalis of Meynert(NBM). Behavioral tests by eight-arm radial maze were conducted 8 weeks, and immunohistochemical staining of choline acetyltransferase(ChAT), serotonin(5-HT), GAD(GABA), amyloid-?protein (AP) was conducted 12 weeks after NBM lesioning. Results In NBM lesioned mice, the ChAT-positive neurons, serotonin-positive neurons, and GAD-positive neurons in right NBM reduced, and ChAT-positive neurons reduced most evidently. At the same time, the ChAT-positive fibers in prefrontal and parietal cortices decreased significantly, serotonin-positive axons slightly, accompanied by heavily AP co-expression. On the contrary, there was no change of GAD-positive neurons in cortex. The working memory error increased significantly.Conclusion Ibotenic acid lesioning in NBM can provide as a model of AD in that it produces deafferentation of cholinergic system and recent memory disruption.
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The present study aims to isolate neural stem cells from neonatal rat hippocampus and induce them to differentiate into cholinergic neurons. A multipotent cell line derived from the hippocampi of neonatal rats which had the ability to form clones was incubated in serum-free DMEM/F12 medium added with 20ng/ml basic fibroblast growth factor (bFGF) and B27. After differentiation of the neural stem cells, immunocytochemistry was used to detect nestin, the antigen of the cell clone, and β-tubulin (Tuj 1 ), glial fibrillary acidic protein (GFAP) and galactocerebroside (Galc), the markers specific for neurons, astrocytes and oligodendrocytes, respectively. Embryonic chick skeletal muscle extract was used to induce the differentiation of the neural stem cells into cholinergic neurons. The results showed that the cell line isolated from the hippocampi of neonatal rats expressed nestin and had the potential to form clones and differentiate into neurons, astrocytes and oligodendrocytes. Embryonic chick skeletal muscle extract can induce 9.6% of the isolated cell line to differentiate into cholinergic neurons compared with 3.9% in controls. These findings suggested that the cell line, which expressed nestin antigen, was a multipotent cell line capable of self-renewing, and was believed to contain stem cells of the CNS. These neural stem cells can be induced to differentiate into cholinergic neurons by using embryonic chick skeletal muscle extract.
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Nerve growth factor (NGF), one of the most potent growth factors for cholinergic neurons, has generated great interest as a potential target for the treatment of Alzheimers disease (AD). The degeneration of basal forebrain cholinergic neurons, which provides the major source of cholinergic innervation to the cerebral cortex and hippocampus, occurs early and contributes significantly to cognitive decline in AD. Those regions show high level expression of NGF and NGF receptors and depend on NGF for their survival and proper function. NGF executes its effects mainly by binding high affinity receptor TrkA in the remaining neurons of AD. Meanwhile, stimulation of neurons may protect those cells from the deleterious effects of AD, a phenomenon called “use it or lose it.”However, the use of NGF as therapeutic agent is limited by their hindered mobility through the blood brain barrier. Many theoretical and technical issues for NGF delivery to the target region in the brain remain to be solved, before NGF can live up to its potential for the treatment of AD.
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Objective To investigate the cholinergic neuron properties and the changes of proliferation and apoptosis of PC 12 cell induced by different concentrations of all trans retinoic acid(RA). Methods PC 12 cell was induced by RA of concentrations of 1-50 ?mol/L. Changes of cell growth curve and cycle, apoptosis and changes of activities of acetylcholinesterase(AchE) and choline acyltransferase(ChAT) were detected by MTT assay, flow cytometry, TUNEL method, spectrophotometry and radiochemistry assay, respectively. Results ① RA significantly enhanced the activity of ChAT with concentrations between 1 to 20 ?mol/L. The activity of ChAT with concentration of 10 ?mol/L was higher than those with other concentrations. ② The apoptosis of RA induced PC 12 cells was markedly enhanced when concentration was higher than 10 ?mol/L. ③ A concentration dependent inhibition of proliferation was demonstrated in the RA induced PC 12 cells. ④ PC 12 cells proliferation was blocked in the G 1→S phase. Conclusion RA can be used to induce PC 12 cells to show the properties of cholinergic neuron with an optimal concentration of 10 ?mol/L.
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Objective To study the correlation between cognitive dysfunction and cholinergic neuron changes in basal forebrain(BFB)and brainstem reticular formation(BSRF)areas after brain con- cussion(BC)in rats.Methods Eighty-four Spragne-Dawley rats weighing 250-310g were used.The BC rat models were reproduced by a self-made simple pendulum impact device,then the rats were ran- domized into one control group and six experimental groups(1 day,2 day,4 day,8 day,16 day,and 24 day groups;n=12 in each group).A Morris Water Maze(MWM)test was used to assess learning and memory function of the rats.Cholinergic neurons in the BFB and BSRF were identified with choline acetyltransferase(CHAT)antibody and quantitated.Results Compared with the control group,the la- tency to find the platform in MWM was much longer on days 1-3 after BC,but there was no statistical difference on days 4-21 after BC.The cell number and the ChAT expression activity of cholinergic neu- rons in the BFB were obviously decreased after BC,and reached the lowest level at 8 days after BC,then increased gradually and nearly reached the normal level at 24 days.The ChAT expression activity in BSRF declined on the first 2 days after BC,then went up gradually,and reached the peak at the 24th day.Conclusion The spatial cognition deficit of BC rats can be detected by MWM in the early period (1-3 days after BC).There are significant changes in the number and ChAT expression activity in BFB and BSRF after BC.The change of cholinergic neurons may be correlated with cognitive deficits in BC rats.
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The cholinergic neurons in the striatal complex are the major interneurons that integrate the informations incoming to and outflowing from the striatum. The shape of synapses may change even after birth and the synaptic morphology reflects the functional state of synapse. However, it is not well known about the synaptic morphology of the mouse striatal cholinergic neurons in their early postnatal life. Thus, we investigated the synaptic morphology of the mouse striatal cholinergic neurons in their early postnatal life by the electron microscopy combined with immunohis-tochemistry. In addition, we investigated the trends of change in synaptic morphology and whether the difference between two compartments exists or not. Experimental animals which are ICR mice, were divided into 5 groups according to their postnatal age: 3-day, 1-week, 2-week, 4-week, and 6-week. Pre-embedding immunohisto-chemistry was done with anti-choline acetyl transferase antibody. The results were as follows. 1. In synapses that immunoreactive terminals constitute the presynaptic components, most of synapses are symmetric type in all age groups (p<0.05). Most of synapses in the dorsal striatum are symmetric form from 1-week of postnatal age, but it is not prominent in the ventral striatum until 2-week of postnatal age. 2. In synapses that immunoreactive terminals constitute the postsynaptic components, both symmetric and asymmetric synapses are noted in similar proportions (p<0.05). There are no difference in the synaptic morphology between dorsal and ventral striatum. 3. No specific findings are observed in synaptic curve according to the postnatal age or compartment. In conclusion, the synaptic morphology of mouse striatal cholinergic neurons is similar to mature pattern from 2-week of postnatal age. And it is thought that period between birth and 2-week of postnatal age is the critical period for synaptogenesis. The synaptic curve does not reflect the degree of synaptic maturity. Further investigations will be required to generalize the synaptic curve as a marker for synaptic maturity.
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Animals , Humans , Mice , Basal Ganglia , Cholinergic Neurons , Critical Period, Psychological , Immunohistochemistry , Interneurons , Mice, Inbred ICR , Microscopy, Electron , Parturition , Synapses , TransferasesABSTRACT
The relationships between cholinergic neurons and SP terminals were examined in the rat sacral ventral horn at the light and electron microscopic levels by means of double immunostaining methods. Cholinergic neurons were labeled by a monoclonal antibody to choline acetyltransferase (CHAT) with the avidin-biotin technique and stained bluish-green by indolyl-?-galactoside reaction products with ?-galactosidase as a marker. On the same sections, SP fibers were labeled by polyclonal antisera to SP after application of the peroxidase-antiperoxidase (PAP) method and stained brown by diaminobenzidine (DAB)reaction. At the light microscopic level CHAT-I neurons stained bluish-green and SP- I fibers Stained brown were found in the ventral horn. At the electron microscopic level, many asymmetrical axodendritic synapses(type I of Gray)were observed between CHAT-I dendrites and SP-I terminals in the ventral horn, but axosomatic synapses and symmetrical synapses (type I of Gray) were hardly detected. These results indicate that SP-I terminals make direct synapses with CHAT-I motoneurons of sacral ventral horn. These synapses may be predominantly excitatory and have importance in the control of muscular constriction.
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Ten to twenty ?l of 20-50% horseradish peroxidase (HRP) in normal saline solution was injected into the myocardium of the anterior wall of left and right ventricles in 11 rabbits. The areas containing sino-atria as well as atrio-ventricular nodes and the dorsal wall of the atria were removed separately. Frozen or cryostate sections of these tissues 25-40?m thick, were incubated according to the HRP (tetramethylbenzidine "TMB" method)-AChE combined method. The blue granules produced by the HRP with TMB were observed in the perikarya of a few AChE intensely positive ganglion cells which were located in the epicardium of the dorsal wall of the atria. This indicated the processes of the HRP-AChE double labeled cells distributed to the myocardium of the anterior wall of the ventriculum. It is generally believed that the nerve cells of strong AChE activity can be referred as cholinergic neurons. For this reason, the present study may give further identification to assert the parasympathetic innervation of the ventricular myocardium. The parasympathetic postganglionic neurons innervated the anterior wall of left and right ventriculi are situated primarily in the epicardium of the posterior wall of the atria.
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Distribution and morphologic features of AChE-containing neurons were observed by the technique of AChE regeneration. There are three kinds: AChE-staining cells--heavily stained cells, medium stained cells and lightly stained cells. Most heavily stained cells are larger multipolar cells. They are located mainly in striatum, basal forebrain, hypothalamus, substantia nigra, locus coeruleus, red nucleus, ventral tegmental nucleus, parabrachial nucleus, pontine tegmental nucleus and the motor nuclei of cranial nerves. The results of AChE-staining were compared with the date of ChAT immunohistochemistry. The relationship between AChE and cholinergic neurons as well as the nature of AChE-containing neurons were discussed.