Computerized analysis of the acetylcholinesterase distribution in the rabbit hippocampal region

Summary The distribution of acetylcholinesterase (AChE) was analysed in the hippocampal region of the rabbit, employing computerized optical densitometry (COD). AChE was demonstrated histochemically according to a modification of the copper thiocholine method, and computerized analysis was performed on a selected section with the use of a graphic operating processor, resulting in images with pixel values ranging between 0 and 255, measuring 512 × 512 pixels. Examples of the densitometric analysis include presentation of pixel values in single points, in profiles through selected areas and in slicing procedures. The results of the densitometric analysis basically agreed with the subjective visual impression of different staining intensities in the sections and corresponding photomicrographs. However, the densitometric analysis provided an objective and more exact expression of the relative AChE content of the different subfields and layers of the hippocampal region. In particular, zones with the same enzyme content as well as zones differing only minimally in activity can be recognized easily and unequivocally. In view of this, the promising future uses of COD are considered briefly.     

银杏内酯对胚基底前脑NOS、AChE阳性神经元发育的影响

目的 探讨银杏内酯对胚基底前脑NOS和AChE阳性神经元发育的影响。方法 实验分成银杏组,NGF组,BDNF组和单纯对照组。取孕17dSD大鼠胚基底前脑原基制成细胞悬液接种于96孔培养板和2块24孔培养板中,分别加入含银杏内酯,NGF,BDNF及不含上述成份的DMEM培养液,于体外培养18d后,96孔培养板行MTT比色分析测定光吸收值（OD值）,以检测培养的神经元活力,2块24孔培养板分别行NADPH-d和AChE组化染色,显微镜下计数各组每了忆中的NOS和AChE阳性神经元数,并用CMM-301图像分析系统对两种神经元的细胞面积和细胞周长进行处理。数据用方差分析和SNK检验进行统计学处理。结果 银杏组MTT比色分析的OD值和NOS,AChE阳性神经元数,细胞面积,细胞周长等指标均明显地好于单纯对照组,达到或仅稍差于NGF组或BDNF组的指标。结论 银杏内酯在促进胚基底前脑NOS和AChE阳性神经元发育方面具有类似NGF和BDNF的作用。     

Differential expression of acetylcholinesterase in the brainstem, ventrobasal thalamus and primary somatosensory cortex of perinatal rats, mice, and hamsters

Acetylcholinesterase (AChE) is transiently expressed by thalamocortical axons in the rat, and staining for this enzyme has been used extensively to study the development of thalamocortical projections. In the present study, patterns of AChE staining were compared in the trigeminal brainstem, thalami and primary somatosensory cortices of perinatal rats, mice, and hamsters. As previously reported, the ventral posteromedial nucleus (VPM) of rats showed dense AChE staining from P-0 at least through P-8. The ventral posterolateral nucleus (VPL) contained heavy AChE staining at least through P-60. In the cortex, there was also dense AChE staining which was organized somatotopically in patches similar to those observed with other methods such as cytochrome oxidase (CO) staining. However, by adulthood, AChE staining revealed a negative image of the CO staining pattern in lamina IV. In the mouse and hamster, there was dense AChE staining inVPL from P-0 through adulthood, but VPM was much less heavily stained for this enzyme. Moreover, the staining in VPL of mice was markedly reduced after transection of axons that travel to the thalamus in the medial lemniscus, suggesting that much of it was contained in these afferent fibers. In the cortices of both perinatal and adult mice and hamsters, AChE staining yielded a negative image of the somatotopically organized patches demonstrable with CO staining. This negative image was apparent by P-2 in the mouse and P-4 in the hamster. These results document a dramatic species difference with respect to the expression of AChE in the thalami and cortices of developing rodents. The differences between the patterns observed in rats vs mice and hamsters probably reflect the fact that cortical AChE in the latter species is not contained in thalamocortical afferents arising from either VPM or VPL.     

Structural differences of pale and strongly stained motor end-plates of the rat diaphragm.

According to the staining intensities for AChE, the motor end-plates of the rat diaphragm can be classified into strong (S) and pale (P) types. About 34% of the total end-plates of the rat diaphragm are of S type and 50% of P type. The P end-plates differ from S end-plates in two aspects. First, the secondary subneural clefts of the S end-plates are well developed. They are numerous, long, closely packed and often branched. On the other hand, the secondary subneural clefts of the P end-plates are short, sparse and usually unbranched. Secondly, there seems to be a variation in AChE activity in the P end-plates. Focal negative AChE areas are found in the subneural apparatus of some P end-plates. It is concluded that the less well developed secondary subneural clefts and focal areas of negative AChE activity contribute to the paler staining of the P end-plates.     

Induction of acetylcholinesterase expression during apoptosis in various cell types

Acetylcholinesterase (AChE) plays a key role in terminating neurotransmission at cholinergic synapses. AChE is also found in tissues devoid of cholinergic responses, indicating potential functions beyond neurotransmission. It has been suggested that AChE may participate in development, differentiation, and pathogenic processes such as Alzheimer's disease and tumorigenesis. We examined AChE expression in a number of cell lines upon induction of apoptosis by various stimuli. AChE is induced in all apoptotic cells examined as determined by cytochemical staining, immunological analysis, affinity chromatography purification, and molecular cloning. The AChE protein was found in the cytoplasm at the initiation of apoptosis and then in the nucleus or apoptotic bodies upon commitment to cell death. Sequence analysis revealed that AChE expressed in apoptotic cells is identical to the synapse type AChE. Pharmacological inhibitors of AChE prevented apoptosis. Furthermore, blocking the expression of AChE with antisense inhibited apoptosis. Therefore, our studies demonstrate that AChE is potentially a marker and a regulator of apoptosis.     

Presynaptic or postsynaptic origin of acetylcholinesterase at neuromuscular junctions? An immunological study in heterologous nerve-muscle cultures

Numerous studies have shown that the acetylcholine receptor (AChR) is inserted in the plasma membrane of the muscle fiber, and that it is focalized at the site of neuromuscular junctions, as an effect of neural influence. In contrast, acetylcholinesterase (AChE) may be presynaptic or anchored in the basal lamina, as well as postsynaptic at neuromuscular junctions. We investigated the origin of the junctional enzyme, particularly the collagen-tailed asymmetric A12 forms, by studying the AChE contents of heterologous rat and chicken neuromuscular cocultures by immunohistochemical and biochemical methods. We found that the overall content of AChE, in the neuromuscular cocultures, including the A12 form, was essentially identical to the sum of the contents of separate myotube and motoneuron cultures. The sedimentation coefficients of the rat and chicken asymmetric forms are sufficiently different to clearly differentiate these enzymes in sucrose gradients: 16 S for rat, 20 S for chicken A12 AChE. Sedimentation analyses of AChE in cocultures thus showed that the A12 form was of muscular origin. In the case of aneural cultures of myotubes, histochemical staining of AChE activity or immunohistochemical staining with specific antibodies showed only very scarce, faint concentrations of enzyme. Some patches of acetylcholine receptor (AChR) were, however, visible in these cultures. Neuromuscular contacts are readily established in cocultures of myotubes with embryonic motoneurons from spinal cords. In the presence of motoneurons, the myotubes presented a larger number of AChR patches. The most remarkable feature of neuromuscular cocultures was the presence of numerous intense AChE patches which always coincided with AChR clusters. By specifically staining nerve terminals with tetanus toxin, we could show an excellent correlation between neuromuscular contacts and the presence of AChE-AChR patches. We found that the AChE patches in heterologous cocultures could be stained exclusively by the anti-myotube AChE antiserum. The focalized enzyme is therefore exclusively, or very predominantly, provided by the myotube.     

Bidirectional axonal transport of 16S acetylcholinesterase in rat sciatic nerve

Axonal transport of the 16S Molecular form of acetylcholinesterase (16S-AChE) in doubly ligated rat sciatic nerves was studied by means of velocity sedimentation analysis on sucrose gradients. This form of AChE was selectively confined to motor, and not to sensory, fibers in the sciatic nerve, where it represented 3--4% of total AChE. Its activity increased linearly with time (4--20 hr) in nerve segments (7 mm) proximal to the central ligature (4.5 mU/24hr) and distal to the peripheral ligature (2.0 mU/24 hr). From the linear rates of accumulation of 16S-AChE, we conclude that the enzyme is conveyed by anterograde and retrograde axonal transport at velocities close to those previously defined for the movement of total AChE (410 mm/day, anterograde; 220 mm/day, retrograde). The transport of AChE molecular forms, other than the 16S form, could not be resolved presumably due to their presence in blood as well as at extraaxonal sites. The present findings are consistent with the view that in rat sciatic nerve most, if not all, of the small portion of total AChE (approximately 3%) which is transported may be accounted for by 16S-AChE.     

Postnatal development of nitrergic and cholinergic structures in rat spinal cord

Nitric oxide (NO) is known to be a freely diffusible gaseous neurotransmitter that is not requiring synaptic connection to exert its effects. Nitric oxide synthase (NOS), the enzyme responsible for NO synthesis can be visualised by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. Other neurotransmitter is a classical neurotransmitter acetylcholine (ACh), regulated by enzyme acetylcholinesterase (AChE) that hydrolyses the acetylcholine after its releasing. This work is presenting results of histochemical study of the NADPH-d and AChE expression (nitrergic and cholinergic neurons) in the spinal cord (SC) during various periods in its development. Specimens from Wistar rat pups in the age ranging from 1st to 21st postnatal days (P1-P21) have been compared with those of adult rats (P90). Transverse sections of the SC were evaluated by light microscope. In adults, the NADPH-d positivity was detectable in the neurons of superficial and deep layers of the dorsal horn, pericentral area and in the area of preganglionic autonomic nuclei. AChE positive structures were seen in the same locations as previous ones with the exception of two locations: in superficial layers of the dorsal horn AChE staining was absent, while in the ventral horn the groups of AChE positive motoneurons were found. At the perinatal period both NADPH-d and AChE positive neurons were stained from slight to moderate intensity only. During later developmental periods the staining gradually increased and achieved adult level of intensity on the day P21. Our results confirmed the presence of nitrergic and cholinergic neurons in investigated areas of the SC and indicated their fully functioning of NADPH-d and AChE positive structures in SC from the third postnatal week.     

Localization of acetylcholinesterase in dissociated cell cultures of the carotid body of the rat

Summary The localization of acetylcholinesterase (AChE) was investigated at the cellular and subcellular levels in dissociated cell cultures of the carotid body of the neonatal rat, prepared by the methods of Fishman and Schaffner (1984). In the presence of iso-OMPA, which blocks non-specific cholinesterase, staining was confined almost exclusively to glomus-cell clusters and occasional isolated cells. These clusters grow as discrete islands scattered throughout the culture and display typical catecholamine (CA) fluorescence as in vivo. AChE staining was abolished or reduced by the cholinesterase inhibitors eserine (30–100 M), or (the poorly lipid soluble) echothiophate (8 (M). Processing of the same culture sequentially for the demonstration of both AChE and CA revealed that glomus-cell clusters and individual glomus cells were consistently positive for both. In electron micrographs AChE reaction product was associated intracellularly with the nuclear envelope and cytoplasm of glomus cells (identified by their characteristic dense cored granules), as well as extracellularly with the boundaries of contiguous glomus cells. Significantly, reaction product occurred in some glomus cell profiles that had both dense-cored and clear (cholinergic-like) vesicles. These findings are discussed in the context of a possible dual (adrenergic/cholinergic) function status of glomus cells in the rat's carotid body.     

Quantitative autoradiographic analysis of muscarinic receptors and quantitative histochemistry of acetylcholinesterase in the rat brain after trimethyltin intoxication

The influence of trimethyl tin (TMT) intoxication on muscarinic cholinergic receptors and histochemistry of acetylcholinesterase (AChE) in the rat brain 21 days after treatment was studied. The topographical distribution and reduction in muscarinic receptor sites were analysed by means of quantitative receptor autoradiography using [³H]quinuclidinyl benzilate (QNB). TMT treatment produced a decrease in cholinergic receptors in a large number of brain regions.

The quantitative distribution of AChE was examined in over 60 regions following TMT intoxication. The activity of AChE was significantly affected. Reduced AChE content was found in several brain regions following TMT intoxication. The effect on AChE content was confined to cholinergic terminal areas, e.g. the hippocampus, while in the area dentata a significant increase in AChE content was detected.

The results are interpreted in terms of TMT producing disruption of the cholinergic system with implications for a neuroanatomical basis of impaired memory mechanisms.     

Effects of Ginkgolide on the development of NOS and AChE positive neurons in the embryonic basal forebrain

Extract of Ginkgo biloba (EGb) has been therapeutically used for several decades to increase peripheral and cerebral blood flow so as to prevent cardiovascular and neurovascular diseases. However, the role of EGb in neuroprotective effects has received much attention recently. In this study, we investigated the effect of EGb on the development of NOS and AChE positive neurons in the rat embryonic basal forebrain. The results showed that treated with EGb, the OD of MTT staining analysis, and the numbers, the cell sizes and circumferences of NOS and AChE positive neurons were greatly promoted. These data suggest that EGb had similar effects of the neurotrophins such as NGF and BDNF in promoting the development of NOS and AChE positive neurons in the rat embryonic basal forebrain.     

Localization of acetylcholinesterase and choline acetyltransferase in the rat pituitary gland

Summary Experiments were conducted to determine the presence of two cholinergic biomarkers, acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) in the rat pituitary. A histochemical procedure for AChE was used to provide visualization of structures containing this enzyme. Radiochemical methods provided a sensitive assay for measuring ChAT activity. Nerve fibres staining for AChE activity were observed in the neurointermediate lobe, with the greatest concentrations appearing at the junction region with the pituitary stalk. Cells staining for AChE were found in the pars distalis and pars intermedia. ChAT activity correlated well with AChE distribution in pars nervosa and pars intermedia but not in pars distalis. The greatest levels of ChAT activity were in pars intermedia and the region where the stalk joins the pituitary. Significant values were also found for the pars nervosa. The presence of AChE and ChAT in pars intermedia and pars nervosa is evidence for a cholinergic innervation to these regions. In pars distalis, where other investigators have found muscarinic receptors, intense staining for AChE and absence of ChAT activity may indicate non-innervated, acetylcholine-sensitive sites.     

Reduced laminin immunoreactivity in the blood vessel wall of ageing rats correlates with reduced innervation in vivo and following transplantation

Changes in extracellular matrix composition and/or organisation, and in particular in the ratio of axonal growth-promoting components such as laminin to growth-inhibiting molecules, could contribute to the degenerative changes observed in the innervation of some peripheral tissues in old age. We have investigated this issue by evaluating laminin content or accessibility at various locations on blood vessels where we had previously studied age-related alterations in innervation density. We have employed a morphological approach, measuring laminin immunoreactivity by a densitometric application of confocal microscopy, because more conventional biochemical techniques would have been unable to distinguish specific, localized changes in laminin at sites accessible to nerves from heterogeneous changes in other areas of the vessel wall, such as the endothelial basal lamina. We found that in 24-month-old rats laminin immunoreactivity is decreased by 50% at the medial-adventitial border in association with the outer layer of smooth muscle cells, where a parallel decrease is observed in innervation density. Axonal terminals were shown to have access to laminin in this region of the blood vessel wall by double staining with laminin and a general neuronal marker. Changes in laminin immunore-activity were region-specific on the same blood vessel, thus excluding the possibility of a generalized decrease in immunoreactivity in old age. For example, in the basilar artery intensity of laminin immunoreactivity decreased in old age at the medial-adventitial border, but showed no change in endothelial cell basal lamina and in the adventitia. Moreover, we performed in oculo transplants of blood vessels displaying differences in laminin immunoreactivity and found that the density of innervation correlated with the intensity of laminin staining, thus lending further support to the hypothesis that laminin might play a role in nerve fibre atrophy in old age.     

大鼠回肠肌间神经丛一氧化氮合酶和乙酰胆碱酯酶双重显示法研究

本文用一氧化氮合酶和乙酰胆碱酯酶双重显示法,对大鼠回肠肌间神经丛进行了组织化学观察,结果发现三种不同染色的神经元：（１）乙酰胆碱酯酶阳性神经元（占８２％）;（２）一氧化氮合酶阳性神经元（占１６％）;（３）一氧化氮合酶和乙酰胆碱酯酶阳性神经元（占２％）。以上结果提示,一氧化氮可以与乙酰胆碱共存于大鼠回肠肌间神经丛的少数神经元内。本文还对肠肌间神经丛内神经元的类型和一氧化氮的作用进行了讨论。     

Location of acetylcholinesterase in axotomized ciliary ganglia

In the present note we have investigated the cytochemical localization of acetylcholinesterase (AChE) in the chick ciliary ganglion (CG) after post-ganglionic axotomy obtained by ablation of the eyeball. Preliminary results show at quite early stages after axotomy a remarkable reduction of cytoplasmic AChE, the residual one being localized in the rough endoplasmic reticulum. On the contrary synaptic areas, in particular those concerning the calyciform synapses, still show a marked AchE activity, similarly to what observed in physiological conditions. The decrease of cytoplasmic AChe in axotomized CG does suggest the possibility that such AChE undergoes to a topographical rearrangement moving towards the synaptic areas of ganglionic neurons.     

Attempted direct visualization of negatively stained amplified immune complex of synaptic acetylcholinesterase using cryoultramicrotomy sections

Summary An immunocytochemical method is proposed for the localization of synaptic acetylcholinesterase (AChE) on ultrathin frozen sections of the electric organ of the electric eel. The immune complex formed is amplified by a non-specific sandwich technique and visualized by negative staining. Definite white spots on synaptic cleft seem to correspond to basal lamina AChE molecules.     

Junctional form of acetylcholinesterase restored at nerve-free endplates.

Rat soleus muscles were ectopically innervated by implanting a foreign nerve in an endplate-free region of muscle and, 2–3 weeks later, cutting the original nerve. The junctional, 16 S form of acetylcholinesterase (AChE) and focal staining for AChE disappeared from the old endplate region within a few days after denervation. In muscles with an ectopic nerve, but not in paired control muscles, 16 S AChE and focal staining were restored in the old endplate region 1–2 weeks after denervation even though nerve fibers could not be detected in that region. These results suggest that the nerve exerts a local effect, specifying the site at which junctional AChE appears, and a nonlocal effect, perhaps mediated by muscle activity, regulating the amount of junctional AChE.     

Age-related changes in acetylcholinesterase and its molecular forms in various brain areas of rats

A previous study conducted in this laboratory revealed a decrease in total cholinesterase (total ChE) in the cerebral cortex, hippocampus and striatum in aged rats (24 months) of various strains, as compared with young animals (3 months). The purpose of the present experiments was to extend the study to other brain areas (hypothalamus, medulla-pons and cerebellum) and to assess whether this decrease was dependent on the reduction of either specific acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE) or both. By using ultracentrifugation on a sucrose gradient, the molecular forms of AChE were evaluated in all the brain areas of young and aged Sprague-Dawley rats. In young rats the regional distribution of total ChE and AChE varied considerably with respect to BuChE. The age-related loss of total ChE was seen in all areas. Although there was a reduction of AChE and, to somewhat lesser extent, of BuChE in the cerebral cortex, hippocampus, striatum, and hypothalamus (but not in the medulla-pons or the cerebellum), the ratio AChE/BuChE was not substantially modified by age. Two molecular forms of AChE, namely G4 (globular tetrameric) and G1 (monomeric), were detected in all the brain areas. Their distribution, expressed as G4/G1 ratio, varied in young rats from about 7.5 for the striatum to about 2.0 for the medulla-pons and cerebellum. The age-related changes consisted in a significant and selective loss of the enzymatic activity of G4 forms in the cerebral cortex, hippocampus, striatum, and hypothalamus, which resulted in a significant decrease of the G4/G1 ratio. No such changes were found in the medullapons or the cerebellum. Since G4 forms have been proposed to be present presynaptically, their age-related loss in those brain areas where acetylcholine plays an important role in neurotransmission may indicate an impairment of presynaptic mechanisms.     

Neurotransmitter Changes in Guinea-Pig Brain Regions Following Soman Intoxication

The effects of the organophosphate acetylcholinesterase (AChE) inhibitor soman (31.2 micrograms/kg s.c.) on guinea-pig brain AChE, transmitter, and metabolite levels were investigated. Concentrations of acetylcholine (ACh) and choline (Ch), noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5-HT), and their metabolites, and six putative amino acid transmitters were determined concurrently in six brain regions. The brain AChE activity was maximally inhibited by 90%. The ACh content was elevated in most brain areas by 15 min, remaining at this level throughout the study. This increase reached statistical significance in the cortex, hippocampus, and striatum. The Ch level was significantly elevated in most areas by 60-120 min. In all regions, levels of NA were reduced, and levels of DA were maintained, but those of its metabolites increased. 5-HT levels were unchanged, but those of its metabolites showed a small increase. Changes in levels of amino acids were restricted to those areas where ACh levels were significantly raised: Aspartate levels fell, whereas gamma-aminobutyric acid levels rose. These findings are consistent with an initial increase in ACh content, resulting in secondary changes in DA and 5-HT turnover and release of NA and excitatory and inhibitory amino acid transmitters. This study can be used as a basis to investigate the effect of toxic agents and their treatments on the different transmitter systems.