Distribution and characteristics of nerve growth factor binding on cholinergic neurons of rat and monkey forebrain

In sections of rat forebrain, perikarya labeled radioautographically with¹²⁵I-NGF resembled cholinesterase-positive neurons in their distribution within striatum and basal forebrain. Neurons with NGF receptors were also visualized in radioautographs prepared from the basal forebrain of a cerebrus monkey. Present techniques fail to detect axons projecting from basal forebrain to hippocampus or cortex which have been shown to take up NGF selectively in retrograde transport studies. In studies with membrane-enriched preparations from rat, high-affinity binding of¹²⁵I-NGF (half maximal saturation in the 15–30 pM range) was detected in basal forebrain and striatum; lower levels of high-affinity binding were seen in hippocampus and neocortex. The binding and molecular properties of these receptors are similar to those described in other NGF-responsive tissues. These observations are further evidence supporting a biological role for NGF on some forebrain cholinergic neurons in adult rat.Special issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

NGF effects on developing forebrain cholinergic neurons are regionally specific

Nerve growth factor (NGF) has been shown to have an effect on neurons in the central nervous system (CNS). A number of observations suggest that NGF acts as a trophic factor for cholinergic neurons of the basal forebrain and the caudate-putamen. We sought to further characterize the CNS actions of NGF by examining its effect on choline acetyltransferase (ChAT) activity in the cell bodies and fibers of developing neurons of the septum and caudate-putamen. ChAT activity was increased after even a single NGF injection. Interestingly, the magnitude of the effect of multiple NGF injections suggested that repeated treatments may augment NGF actions on these neurons. The time-course of the response to NGF was followed after a single injection on postnatal day (PD) 2. NGF treatment produced long-lasting increases in ChAT activity in septum, hippocampus and caudate-putamen. The response in cell body regions (septum, caudate-putamen) was characterized by an initial lag period of approximately 24 hr, a rapid rise to maximum values, a plateau phase and a return to baseline. The response in hippocampus was delayed by 48 hr relative to that in septum, indicating that NGF actions on ChAT were first registered in septal cell bodies. Finally, developmental events were shown to have a regionally specific influence on the response of neurons to NGF. For though the septal response to a single NGF injection was undiminished well into the third postnatal week, little or no response was detected in caudate-putamen at that time. In highlighting the potency and regional specificity of NGF effects, these observations provide additional, support for the hypothesis that NGF is a trophic factor for CNS cholinergic neurons.Dedicated to Dr. E. M. Shooter and Dr. S. Varon as part of a special issue (Neurochemical Research, Vol. 12, No. 10, 1987).     

Localization and quantity of hyaluronan in urogenital organs of male and female rats

The histochemical distribution of hyaluronan was analysed in various urogenital organs of male and female (non-pregnant and pregnant) rats by use of a hyaluronan-binding protein and avidin biotin/peroxidase staining. Microwave-aided fixation was used to preserve the extracellular location of hyaluronan. The concentrations of hyaluronan in the different tissues were measured with a highly sensitive radio-assay. Hyaluronan accumulated predominantly in the connective tissue around smooth muscle fibres and in the subepithelial lamina propria. Abundant hyaluronan also occurred in perivascular and perineural connective tissue. In the female urogenital organs, hyaluronan content was high in the vagina and urinary bladder, and highest in the vagina during pregnancy. In the uterus, the surface epithelium of the endometrium stained intensely. In the ovary, the zona pellucida of the oocyte and the theca interna cell layer of the follicles and the follicular fluid of mature follicles exhibited prominent staining. The corpus luteum was devoid of hyaluronan, whereas enlarged corpora lutea of pregnancy exhibited weak, patchy staining. In male urogenital organs, staining for hyaluronan was absent from the testis and epididymis, whereas the erectile connective tissue of the penis stained intensely. The hyaluronan concentrations were high in penile tissue and urinary bladder, while testis, epididymis and the ductus deferens contained only little hyaluronan.     

Expression of neuronal-NOS in developing basal forebrain cholinergic neurons: Regulation by NGF

Nerve growth factor (NGF) acts through the receptor tyrosine kinase trkA to serve as a trophic factor for cholinergic neurons in the medial septal nucleus and vertical limb of the diagonal band. We have previously shown that the neuronal isoform of nitric oxide synthase (NOS) is selectively expressed in a large fraction of trkA-expressing cholinergic neurons in these brain regions in the adult rat, and that NGF induces the expression of neuronal-NOS in these cells. Herein, we show that: 1) neuronal-NOS is also localized to these neurons in the developing septum; 2) the expression of neuronal-NOS is regulated in the developing medial septal nucleus and vertical limb of the diagonal band; 3) neuronal-NOS regulation parallels that for other markers of basal forebrain cholinergic neuron differentiation, such as cholineacetyltransferase; and 4) NGF infusion in the postnatal period induces robust increases in neuronal-NOS mRNA and in NOS activity in the basal forebrain. Taken together with earlier findings, our results suggest that neuronal-NOS has a role in the differentiation and mature function of septal cholinergic neurons. Through enhancing neuronal-NOS synthesis, endogenous NGF is likely to regulate NO functions in vivo. Special issue dedicated to Dr. Hans Thoenen.     

Regulation of choline acetyltransferase/iacZ fusion gene expression in putative cholinergic neurons of drosophila melanogaster

We have analyzed the distribution of putative cholinergic neurons in whole-mount preparations of adult Drosophila melanogaster. Putative cholinergic neurons were visualized by X-gal staining of P-element transformed flies carrying a fusion gene consisting of 5′ flanking DNA from the choline acetyltransferase (ChAT) gene and a lacZ reporter gene. We have previously demonstrated that cryostat sections of transgenic flies carrying 7.4 kb of ChAT 5′ flanking DNA show reporter gene expression in a pattern essentially similar to the known distribution of ChAT protein. Whole-mount staining of these same flies by X-gal should thus represent the overall distribution of ChAT-positive neurons. Extensive staining was observed in the cephalic, thoracic, and stomodeal ganglia, primary sensory neurons in antenna, maxillary palps, labial palps, leg, wing, and male genitalia. Primary sensory neurons associated with photoreceptors and tactile receptors were not stained. We also examined the effects of partial deletions of the 7.4 kb fragment on reporter gene expression. Deletion of the 7.4 kb fragment to 1.2 kb resulted in a dramatic reduction of X-gal staining in the peripheral nervous system (PNS). This indicates that important regulatory elements for ChAT expression in the PNS exist in the distal region of the 7.4 kb fragment. The distal parts of the 7.4 kb fragment, when fused to a basal heterologous promoter, can independently confer gene expression in subsets of putative cholinergic neurons. With these constructs, however, strong ectopic expression was also observed in several non-neuronal tissues. © 1995 John Wiley & Sons, Inc.     

circRNA Acbd6 promotes neural stem cell differentiation into cholinergic neurons via the miR-320-5p-Osbpl2 axis

Neural stem cells (NSCs) persist in the dentate gyrus of the hippocampus into adulthood and are essential for both neurogenesis and neural circuit integration. Exosomes have also been shown to play vital roles in regulating biological processes of receptor cells as a medium for cell-to-cell communication signaling molecules. The precise molecular mechanisms of exosome-mediated signaling, however, remain largely unknown. Here, we found that exosomes produced by denervated hippocampi following fimbria–fornix transection could promote the differentiation of hippocampal neural precursor cells into cholinergic neurons in coculture with NSCs. Furthermore, we found that 14 circular RNAs (circRNAs) were upregulated in hippocampal exosomes after fimbria–fornix transection using high-throughput RNA-Seq technology. We further characterized the function and mechanism by which the upregulated circRNA Acbd6 (acyl-CoA-binding domain–containing 6) promoted the differentiation of NSCs into cholinergic neurons using RT–quantitative PCR, Western blot, ELISA, flow cytometry, immunohistochemistry, and immunofluorescence assay. By luciferase reporter assay, we demonstrated that circAcbd6 functioned as an endogenous miR-320-5p sponge to inhibit miR-320-5p activity, resulting in increased oxysterol-binding protein–related protein 2 expression with subsequent facilitation of NSC differentiation. Taken together, our results suggest that circAcbd6 promotes differentiation of NSCs into cholinergic neurons via miR-320-5p/oxysterol-binding protein–related protein 2 axis, which contribute important insights to our understanding of how circRNAs regulate neurogenesis.     

Sensory neurons and peripheral pathways in Drosophila embryos

Summary The thoracic and abdominal segments of the Drosophila embryo contain 373 neurons innervating external sensory structures and 162 neurons innervating chordotonal organs. These neurons are arranged in ventral, lateral and dorsal clusters within each segment, in a highly invariant pattern. Two fascicles are formed in each segment as the sensory axons grow ventrally towards the CNS and meet motor axons growing dorsally from the CNS. In all but the last segment, the anterior fascicle is contributed by the dorsal and lateral neurons, while the posterior one is formed by the ventral neurons. Five distinct segmental patterns are described, corresponding to (1) the prothorax, (2) the other two thoracic segments, (3) the first seven abdominal segments, (4) the eighth and (5) the ninth (and possibly the tenth) abdominal segments.The publisher regrets that two companion papers unfortunately were published out of sequence. The present paper should have preceded the paper entitled The sense organs in the Drosophila larva and their relation to the embryonic pattern of sensory neurons, which appeared in Volume 195, Number 4 of the journal (pp 222–228)     

Neuroprotective efficacy of thymoquinone against amyloid beta-induced neurotoxicity in human induced pluripotent stem cell-derived cholinergic neurons

The natural antioxidant Thymoquinone (TQ) is the most abundant ingredient in the curative plant Nigella sativa seed's oil. An extensive number of studies have revealed that TQ is the most active and most responsible component for the plant's pharmacological properties. It has been documented in several studies that TQ has a wide range of protective activities and many neuropharmacological attributes. Amyloid beta (Aβ) is the major role player peptide in the progression of Alzheimer's disease (AD). Our current study has been implemented to explore the protective possibilities of TQ on Aβ_1–42 -induced neurotoxicity. To test TQ's effect we used cultured human induced pluripotent stem cell (hiPSC)-derived cholinergic neurons. The obtained results showed that Aβ_1–42 caused cell death and apoptosis, which was efficiently attenuated by the co-treatment of TQ. Moreover, TQ restored the decrease in the intracellular antioxidant enzyme glutathione levels and inhibited the generation of reactive oxygen species induced by Aβ_1–42. Furthermore, using the fluorescent dye FM1–43 we demonstrated that TQ was able to reduce synaptic toxicity caused by Aβ_1–42. Thus, the findings of our study suggest that TQ holds a neuroprotective potential and could be a promising therapeutic agent to reduce the risk of developing AD and other disorders of the central nervous system.     

Bone morphogenetic proteins (BMP6 and BMP7) enhance the protective effect of neurotrophins on cultured septal cholinergic neurons during hypoglycemia

The effects of two bone morphogenetic proteins (BMP6, BMP7), alone and in combination with neurotrophins, were tested on cultures of embryonic day 15 rat septum. A week-long exposure to BMP6 or BMP7 in the optimal concentration range of 2-5 n M increased the activity of choline acetyltransferase (ChAT) by 1.6-2-fold, in both septal and combined septal-hippocampal cultures. The increase in ChAT activity reached significance after 4 days and continued to increase over an 11-day exposure. Under control culture conditions neither BMP significantly altered the number of cholinergic neurons, and BMP effects on ChAT activity were less than linearly additive with those of nerve growth factor. The effects of BMPs and BMP + neurotrophin combinations were also assayed under two stress conditions: low-density culture and hypoglycemia. In low-density cultures BMPs and BMP + neurotrophin combinations preserved ChAT activity more effectively than neurotrophins alone. During 24 h hypoglycemic stress, BMPs alone did not preserve ChAT activity, but BMP + neurotrophin combinations preserved ChAT activity much more effectively than neurotrophins alone. These results demonstrate that BMP6 and BMP7 enhance ChAT activity under control and low-density stress conditions, and that during a hypoglycemic stress their trophic effect requires and complements that exerted by neurotrophins.     

Indoleamine-accumulating neurons in the retina of rabbit,cat and goldfish

Summary Special neurons accumulating indoleamines have been detected in the retina of rabbit, cat and goldfish. They have their perikarya in the inner-most cell row of the inner nuclear layer, among the amacrine cells, and send their processes to various parts of the inner plexiform layer. The distribution of the processes is different in the different animals investigated. The neurons do not correspond to the previously known dopaminergic retinal neurons, which have a different distribution of their terminals and which can be demonstrated with a specially developed technique, simultaneously with the indoleamine-accumulating neurons.This work was supported by grants from the Swedish Medical Research Council (project 04X-2321), the Medical Faculty of the University of Lund, the Åke Wibergs Stiftelse and the Magn. Bergvalls Stiftelse     

Localization of cholinergic neurons in the cat lower brain stem

The localization of cholinergic neurons in the cat lower brain stem was determined immunocytochemically with a monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme. ChAT-positive neurons were observed in four major cell groups: cranial nerve motor and special visceromotor neurons: parasympathetic preganglionic visceromotor neurons; neurons located in the ponto-mesencephalic tegmentum including area X (or pedunculopontine tegmental nucleus), nucleus laterodorsalis tegmenti (Ldt) of Castaldi, and peri-locus coeruleus alpha (peri-alpha); and neurons located in nucleus reticularis magnocellularis (Mc) and adjacent nucleus reticularis gigantocellularis (Gc) of the medulla.     

Possible axo-axonal synapses between peripheral adrenergic and cholinergic nerve terminals

Summary The relations between adrenergic and cholinergic terminals were studied in rat iris and rat heart with the electron microscope. Adrenergic terminals were identified by treating the animals with 5-hydroxydopamine, which produces dense-cored synaptic vesicles in adrenergic terminals in tissues fixed in glutaraldehyde and osmium. The specificity of this observation was verified. It was found that adrenergic and cholinergic nerve terminals often come in close contact with one another, the distance between the adjoining membranes being about 250 Å. At times, faint membrane thickenings could be observed in these places. The available pharmacological, physiological, and morphological evidence leaves little room for doubt that cholinergic terminal fibres can influence the adrenergic fibres. From mainly morphological evidence, it is also postulated that adrenergic terminals influence cholinergic ones.This work was supported by grants from the United States Public Health Service (06701-04), the Faculty of Medicine, University of Lund, Lund, Sweden and the Swedish Medical Research Council (B70-14X-2321-03 and B70-14X-712-05). Svenska sällskapet för medicinsk forskning.     

Molecularly defined and functionally distinct cholinergic subnetworks

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梭曼对大鼠应激性体温过高的作用及中枢和外周胆碱能受体阻断剂对其效应的影响

目的：观察梭曼对大鼠应激性体温过高的抑制作用以及中枢和外周胆碱能受体阻断剂对其效应的影响。方法：用无线遥测技术测量大鼠的体温,观察皮下注射梭曼、东莨菪碱、甲基东莨菪碱和吡啶斯的明对开放环境中大鼠应激性体温过高的影响。用分光光度技术测定血浆中胆碱酯酶活性。结果：①对照组大鼠在开放实验箱中体温升高达0.96℃,而注射梭曼动物体温只升高了0.55℃。中枢性胆碱能受体阻断剂东莨菪碱几乎完全阻断梭曼对应激体温过高的抑制作用,而外周胆碱能受体阻断剂甲基东莨菪碱则能明显增强梭曼对应激性体温过高的抑制作用。②外周抗胆碱酯酶剂吡啶斯的明能使血浆胆碱酯酶的活性降低至52%,同时明显提高开放环境中大鼠应激性体温过高的反应。甲基东莨菪碱几乎可以阻断吡啶斯的明对应激体温过高反应的影响。结论：神经毒剂梭曼可改变大鼠在开放环境中应激性体温过高的反应能力,其作用主要是通过中枢毒蕈碱型胆碱能通路所致。此外,外周胆碱能神经参与大鼠开放应激性体温过高的形成过程。     

Cells capable of uptake of horseradish peroxidase in some circumventricular organs of the cat and rat

Summary The structure of mesenchymal cells distributed in some of the hypendymal organs of the circumventricular system in the cat and rat was demonstrated after intravenous injection of high doses of horseradish peroxidase. These cellular elements were observed in the vicinity of blood vessels of the organon vasculosum laminae terminalis, subfornical organ and area postrema. Electron-microscopically, these cells located between the basal laminae of the brain parenchyma and the blood capillaries show long cellular processes encircling fenestrated capillaries. Light and electron-microscopic examination revealed that this cell type is identical with the horseradish peroxidase-uptake cells, previously reported in the vicinity of the hypophysial portal system. Such phagocytic cells may be considered as a cellular component intervening between the brain parenchyma and the blood stream, playing a role in selective barrier functions in the above-mentioned circumventricular organs where a blood-brain barrier in the classical sense of the definition is lacking.This work was supported by grant No. 437002 from the Ministry of Education, Science and Culture, Japan     

A comprehensive study on long‐term injury to nigral dopaminergic neurons following intracerebroventricular injection of lipopolysaccharide in rats

Parkinson's disease (PD) is characterized by selective and progressive degeneration of dopaminergic neurons in the substantia nigra (SN). Lipopolysaccharide (LPS) can induce chronic inflammation and has been widely used to study the pathogenesis of PD. In this study, a single intracerebroventricular injection of LPS was used to induce neurotoxic effects on dopaminergic neurons in Sprague–Dawley rats. The long‐term neurotoxic effects of LPS were evaluated at different time points. Microglia were activated in the hippocampus and striatum at 4 weeks, and in the SN at 24 weeks. Astrocytes were activated in the hippocampus and nigrostriatal system at 2 and 24 weeks. The expression of brain‐derived neurotrophic factor in the SN increased at 4 weeks and decreased after 12 weeks, and tyrosine hydroxylase‐positive neurons in the SN were shown to have an atrophic appearance, with cell loss evident after 24 weeks. Phospho‐α‐synuclein expression, a reflection of parkinsonian pathogenesis, increased at 12 weeks, and peaked at 24 weeks. Abnormal motor behavior appeared at 16 weeks and lasted up to 48 weeks. These results indicate that microglia are activated for several months after a single, low dose injection of LPS, which eventually results in progressive and selective damage to dopaminergic neurons in the SN.