Optimal Parameters for the Histochemical Demonstration of Acetylcholinesterase in Plastic Sections of Rat Brain

A modified method for improved preservation and optical resolution of acetylcholinesterase (AChE)-containing structures in adult rat brain is described. Optimal tissue preparation included fixation in paraformaldehyde 4%, glutaraldehyde 0.1%, and sucrose 7% in 0.1 M Sorensen's phosphate buffer, pH 7.4, rinsing in buffer 50 mM with respect to NLL, Q and 2% with respect to sucrose, acetone dehydration, vacuum infiltration widi LKB Historesin, and polymerization at 4 C, overnight incubation of 10 μm sections at 37 C in the AChE histochemical reaction mixture and silver intensification according to Hedreen et al. Demonstration of AChE enzyme activity in the cholinergic projection from the rat basal forebrain to the ipsilateral hippocampus exemplifies the usefulness of the technique. The method provides an excellent demonstration of AChE-positive axonal processes and enables the pharmacohis-tochemical visualization of cholinergic neurons. This procedure offers a convenient method for analysis of cholinergic neurons that avoids potential artifacts inherent in other AChE histochemical procedures.     

A comparison of the localization of acetylcholinesterase in the rat brain as demonstrated by enzyme histochemistry and immunohistochemistry

The localization of acetylcholinesterase (AChE) as revealed either by enzyme-histochemical or by immunohistochemical methods was compared in distinct regions of the rat brain. In general, the localization of AChE observed was nearly the same, whether revealed by histochemical demonstration of its catalytic activity or by immunohistochemical detection of the enzyme molecule itself, in all regions investigated. Penetration problems of the antibodies, however, arose on strong myelin sheaths of the facial nerve, for instance, where no immunohistochemical staining was found though there was a relatively strong histochemical reaction. These problems could be partly solved by increasing the normal concentration of Triton X-100 added to the immunohistochemical solutions (0.1%) to 2.5%. Furthermore, it seems that sites containing low amounts of AChE could be better detected by the enzyme-histochemical method, whereas the depiction of structures (particularly of nerve fibres) was somewhat sharper with the immunohistochemical method.     

Localization of substance P mRNA in cholinergic cells of the rat laterodorsal tegmental nucleus:In situ hybridization histochemistry and immunocytochemistry

1. In situ hybridization histochemical techniques in combination with immunocytochemistry and acetylcholinesterase (AChE) histochemistry were used to study the colocalization of messenger RNA (mRNA) encoding the neuropeptide substance P (SP) in cholinergic cells of the laterodorsal tegmental nucleus (LDT) of the rat pontine brain stem. 2. Alternate serial sections were hybridized with a 48-base, 35S-labeled synthetic oligonucleotide probe encoding SP using in situ hybridization histochemistry and processed either histochemically for AChE or immunocytochemically for choline acetyltransferase (ChAT). 3. In addition, serial section analysis was used to demonstrate the correlation between SP and SP mRNA in the same cells of the LDT. 4. These studies reveal that the cholinergic neurons of the LDT synthesize SP.     

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.     

Effect of preganglionic sympathectomy on the cholinesterase activity in the superior cervical ganglia of rats and hamsters

Summary By employing biochemical assay and histochemical enzyme techniques the effect of preganglionic sympathectomy on the cholinesterase (ChE) activity in the superior cervical ganglia of rats and hamsters was investigated. Biochemical assays indicate that the ChE activity in the superior cervical ganglia of adult rats and hamsters is 57.19 and 28.63 respectively (expressed in u moles acetylcholine hydrolyzed per min per g of tissue); two weeks after preganglionic denervation, about 50% and 60% of ChE activity are lost respectively. Histochemical enzyme examination reveals that in the rat superior cervical ganglion, the majority of the neurons are adrenergic with weak to moderate acetylcholinesterase (AChE) reaction and the minority of the neurons are cholinergic with strong AChE activity, while only one type of adrenergic neurons exhibits a weak AChE activity in the hamster superior cervical ganglion. The AChE activity is localized in the perinuclear area, in the cisternae of the rough surfaced endoplasmic reticulum, in the Golgi complex and on the plasma membrane of the hamster's neurons; it is mainly localized in the cisternae of the rough surfaced endoplasmic reticulum of the rat's neurons. AChE reaction product is also detected on the axolemmal membranes of the preganglionic nerve fibers in the sympathetic ganglia of rats and hamsters.After preganglionic sympathectomy, the AChE activity in the adrenergic neurons and in the preganglionic unmyelinated nerve fibers is markedly reduced, whereas the cholinergic neurons and preganglionic myelinated nerve fibers remain unchanged. On the basis of these results two conclusions have been reached: (1) The fact that strong AChE activity localized in the cholinergic neurons and preganglionic myelinated fibers is not influenced by denervation, suggests that these structures are able to produce AChE. (2) The reduction of AChE activity in the rat and hamster superior cervical ganglia two weeks after preganglionic denervation, observed by histochemical examination, can be correlated with a concomitant measurable reduction determined by biochemical assays.Supported in part by a grant from the National Science Council, Republic of China. The author wishes to express his gratitude to the Department of Pharmacology, College of Medicine, National Taiwan University, for the use of its equipment for biochemical assays     

Cholinesterase in the posterior and intermediate lobes of the pituitary

Summary Posterior and intermediate lobes of pituitary glands of cat, rabbit, beef, and rat were examined histochemically for specific (AChE) and non-specific (BuChE) cholinesterase by light and electron microscopy. Acetylthiocholine was utilized in conjunction with ethopropazine to demonstrate AChE, and butyrylthiocholine with BW 284C51 to demonstrate BuChE. Glandular cells of the intermediate lobe of cat, rabbit and rat contained variable amounts of AChE, whereas those of beef contained BuChE. In the posterior pituitary, AChE was detected in the cat, BuChE in the beef and rat, and both AChE and BuChE in the rabbit. In the posterior lobe of all species examined, cholinesterase, whether true or pseudo enzyme, as the case may be, was localized to certain pituicytes and pituicyte-neuron junctions. These histochemical studies failed to identify cholinergic neurons in the posterior pituitary. Large blood vessels of the pituitary were innervated apparently by adrenergic nerves only. Speculations on the role of pituicyte cholinesterase in posterior pituitary secretion are presented.Supported by the Medical Research Council of Canada.Medical Research Associate of the MRC of Canada.     

The presence of acetylcholinesterase-positive nerve fibres in the deep pineal gland and the pineal stalk but not in the superficial part of adult albino rats

Application of the histochemical method for testing acetylcholinesterase (AChE, EC 3.1.1.7) showed the presence of AChE-positive nerve fibers in the deep pineal gland and the pineal stalk but not in the superficial part of adult albino rats. These findings may indirectly support the existence of the potentially cholinergic innervation of at least some of the rat pinealocytes present in these parts of the gland and augment the evidence of the heterogeneity of the rat pinealocytes. It is possible that cholinergic neurons in the medial habenular nuclei or in the parasympathetic sphenopalatine ganglion may be a source of these AChE-positive fibres. The examination was performed at the light microscope level.     

A comparison of the localization of acetylcholinesterase in the rat brain as demonstrated by enzyme histochemistry and immunohistochemistry

Summary The localization of acetylcholinesterase (AChE) as revealed either by enzyme-histochemical or by immunohistochemical methods was compared in distinct regions of the rat brain. In general, the localization of AChE observed was nearly the same, whether revealed by histochemical demonstration of its catalytic activity or by immunohistochemical detection of the enzyme molecule itsclf, in all regions investigated. Penetration problems of the antibodies, however, arose on strong myelin sheaths of the facial nerve, for instance, where no immunohistochemical staining was found though there was a relatively strong histochemical reaction. These problems could be partly solved by increasing the normal concentration of Triton X-100 added to the immunohistochemical solutions (0.1%) to 2.5%. Furthermore, it seems that sites containing low amounts of AChE could be better detected by the enzymehistochemical method, whereas the depiction of structures (particularly of nerve fibres) was somewhat sharper with the immunohistochemical method.Dedicated to Professor Dr.T.H. Schiebler on the occasion of his 65th birthday     

Immobilization of rat brain acetylcholinesterase on ZnS and poly(indole-5-carboxylic acid) modified Au electrode for detection of organophosphorus insecticides

A novel, highly sensitive amperometric biosensor for detection of organophosphorus (OP) compounds has been constructed, based on rat brain acetylcholinesterase (AChE) immobilized onto nanocomposite of ZnS-nanoparticles (ZnSNPs) and poly(indole-5-carboxylic acid) electrodeposited on Au electrode. In the presence of acetylthiocholine chloride (ATCl) as a substrate, ZnSNPs promoted electron transfer reactions at a lower potential and catalyzed electrochemical oxidation of enzymatically formed thiocholine, thus increasing detection sensitivity. Under optimum conditions (phosphate buffer, pH 7.5 and 30°C), the inhibition of AChE by malathion and chlorpyrifos was proportional to their concentrations in the range, 0.1-50nM and 1.5-40nM, respectively. The biosensor determined malathion and chlorpyrifos in spiked tap water samples with a acceptable accuracy (95-100%). The enzyme electrode had long-storage stability (50% retention of initial activity within 2 months, when stored at 4°C).     

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。     

The multiple forms of brain acetycholinesterase. III. Implications for the histochemical demonstration of acetylcholinesterase

The multiple forms of acetylcholinesterase (AChE, E.C. 3.1.1.7) have been investigated with regard to their histochemical demonstrability. Their pattern is influenced by buffer treatment, fixation, and by incubation conditions causing aggregation and disaggregation as well as loss or inactivation of individual forms. The standard histochemical method for AChE preferentially demonstrates the high molecular forms. Most of the oligomer forms are washed out or inactivated. A selective demonstration of the highly aggregated forms is possible either by inhibition of the oligomers with diisopropylfluoridate (DFP) or by specifically dissolving them out. No reason could be found for the selective demonstration of the low molecular weight forms.     

Developmental changes and acetylcholinesterase activity in the metamorphosing brain of Tenebrio molitor: Correlation to ecdysteroid titers

The brain of Tenebrio molitor exhibited marked fluctuations in acetylcholinesterase (AChE) activity throughout metamorphosis. This was true AChE activity, since it was inhibited by high substrate concentrations and by 10 μM of the specific AChE inhibitor BW284C51 [(1,5-bis'4-allyldimethylammoniumphenyl)-pentan-3-one dibromide] but not by iso-OMPA (tetraisopropylpyrophosphoramide), a cholinesterase (but not AChE) inhibitor. The histochemical AChE activity was localized in the neuropile and the nuclear envelope of neurons and glial cells. The enzyme extracted from brains with 1% Triton X-100 and 1 M NaCl sedimented as a single peak in a sucrose density gradient, with a sedimentation coefficient of 5.4S. This single AChE sedimentation peak was mainly due to an amphiphilic dimeric form. AChE activity per brain increased in newly ecdysed pupa. AChE activity per milligram of protein exhibited a peak in the mid-pupa which could be correlated to the increase in ecdysteroid titers. © 1994 Wiley-Liss, Inc.     

TRANSPORT OF CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN THE RAT SCIATIC NERVE: A BIOCHEMICAL AND ELECTRON HISTOCHEMICAL STUDY

The transport of acetylcholinesterase (AChE) and choline acetyltransferase (ChAc) were investigated by biochemical and histochemical methods. After ligature of one of the sciatic nerves of the rat for varying times—4, 14, 20 and 44 h—the normal levels and the accumulation of AChE and ChAc activities were investigated. It can be inferred from the results that there is a rapid accumulation of AChE activity just proximal to the ligature, while the increase in ChAc activity is less pronounced. Distal to the ligature the level of AChE is above the control value whereas, in contrast to this, the ChAc activity is significantly decreased. Histochemical demonstration of the two enzymes indicates that they are present in the cholinergic axons. The reaction end-product produced by AChE occurs within vesicles and neurotubules, while the endproduct due to ChAc appears to be free in the axoplasm, bound to neurofilaments and on the outer surface of vesicles and tubules.     

Zinc-iodide-osmium procedures as markers of subcellular structures. I. Standardization of staining of transmitter containing vesicles

In the present investigation certain stain properties of the zinc iodide-osmium tetroxide mixture were investigated. It was observed that the type of reaction of certain cell structures with a ZIO mixture largely depended on several factors, namely, the pH of the mixture, aldehyde prefixation and type (s) of buffer (s) used. The standardization of these parameters led to the development of four procedures, each one of them with distinct stain properties. A nomenclature to designate these methods is proposed. The following procedures were applied to material processed for electron microscopy: 1. C.4.4-ZIO-4 degree -18 h: the ZIO mixture was prepared in citric acid-disodium phosphate buffer pH 4.4 and the tissue was incubated at 4 degree C during 18 H; 2. K-P.7.4-C.4.4-ZIO-4 degree -18 h: the tissue was prefixed in Karnovsky fixative prepared in phosphate buffer pH 7.4 and then incubated in C.4.4-ZIO at 4 degree C during 18 h; 3. V.7.4-ZIO-4 degree -18 H: the ZIO was prepared in veronal buffer pH 7.4 and incubation of the tissue was at 4 degree during 18 H; 4.K-P.7.4-V.7.4-ZIO-4 degree -18 h: the tissue was prefixed in Karnovsky fixative prepared in phosphate buffer pH 7.4 and then incubated in V.7.4-ZIO at 4 degree C during 18 h. The chromaffin cells and the cholinergic endings of the rat adrenal medulla and the vas deferens nerves were studied. C.4.4-ZIO-4 degree -18 h: This procedure stained adrenaline and noradrenaline storing granules. Synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted synaptic vesicles at cholinergic endings were not stained. K-P.7.4.4-ZIO-4 degree -18 h: One type of chromaffin granule (probably storing noradrenaline) and both, the small and the granulated synaptic vesicles of cholinergic endings were deeply stained with this method. The aminergic fibres of the vas deferens reacted negatively. V.7.4-ZIO-4 degree -18 H: Both types of chromaffin granules and only the small synaptic vesicles of cholinergic endings were revealed with this procedure. In addition, some compartments of the Golgi complex were also stained. K-P.7.4-V.7.4-ZIO-4 degree -18 h: This method did not stain adrenaline and noradrenaline storing granules. Cholinergic synaptic vesicles appeared stained. However, the most striking stain property of this procedure was the staining of many cell organelles. The probable mechanisms by which different factors affect the ZIO reaction are discussed.     

Ultrastructural localization of arylsulfatase C activity in rat kidney

Metal precipitation techniques for ultrastructural demonstration of arylsulfatase C activity were studied in rat kidney. Possible substrates for the techniques were biochemically tested with regard to their velocity of enzymatic hydrolysis and their specificity for arylsulfatase C. Effects of buffers and capturing metals were also examined. The results of these biochemical studies were then verified histochemically. Incubation in a medium containing 1 mM 4-methylumbelliferyl sulfate, 1% barium chloride, 0.1 M imidazole-HCl buffer (pH 7.5), and 5% sucrose achieved identifiable results in adequately fixed kidney. Precipitation of barium sulfate was localized mainly in the endoplasmic reticulum and perinuclear cisterns of the epithelial cells in the descending portions of proximal tubules.     

Cholinergic pyramidal neurons of the motor region of human neocortex

By means of histochemical methods for revealing +choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) cytoarchitectonic of the field 4 of the motor cortex of the cerebrum has been studied in 5 persons at the age of 33-65 years. An essential part of neurons at revealing AChE and most of them at revealing ChAT do not react. Among giant pyramidal neurons (Bets) according to ChAT activity, 4 types are distinguished: neurons with low, middle, high and very high activity. The presence of ChAT is ascertained in middle and large pyramidal neurons of the III layer. Presence of ChAT-positive synapses is demonstrated in apical dendrites. A conclusion is made that less part of the pyramidal in the III, V layers are cholinergic ones.     

Molecular properties of acetylcholinesterase in mouse spleen

The presence of acetylcholinesterase (AChE) mRNA and activity in the tissues and cells involved in immune responses prompted us to investigate the level and pattern of AChE components in spleen. AChE activity was higher in mouse spleen (0.46 +/- 0.13 micromol of acetylthiocholine split per hour and per mg protein) than in muscle or heart, but lower than in brain. The spleen was essentially free of butyrylcholinesterase (BuChE) activity. About 40% of spleen AChE was extracted with a saline buffer, and a further 40% with 1% Triton X-100. Sedimentation analyses, the splitting of subunits in AChE dimers, phosphatidylinositol-specific phospholipase C (PIPLC) exposure, and phenyl-agarose chromatography showed that hydrophilic (G1H, 43%) and amphiphilic AChE monomers (G1A, 36%), as well as amphiphilic dimers (G2A, 21%), occurred in spleen. All these molecules bound to fasciculin-2-Sepharose, although the extent of binding was higher for G1H (77%) than for G1A (63%) or G2A (48%) forms. Differences in the extent to which wheat germ lectin (WGA) adsorbed with AChE of mouse spleen and of erythrocyte allowed us to discard the blood origin of spleen AChE activity. A 62 kDa protein was labeled in spleen samples using antibodies against human AChE. The protein was attributed to AChE monomers since its size was the same, regardless of whether disulfide bonds were reduced or not. Since cholinergic stimulation modulates proliferation/maturation of lymphoid cells, AChE may be important for regulating the level of acetylcholine (ACh) in the neighborhood of cholinergic receptors (AChR) in spleen and other lymphoid tissues.     

Solubilization of Membrane-Bound Acetyicholinesterase by a Phosphatidylinositol-Specific Phospholipase C

Phosphatidylinositol-specific phospholipase C (PIPLC) quantitatively solubilizes acetylcholinesterase (AChE) from purified synaptic plasma membranes and intact synaptosomes of Torpedo ocellata electric organ. The solubilized AChE migrates as a single peak of sedimentation coefficient 7.0S upon sucrose gradient centrifugation, corresponding to a subunit dimer. The catalytic subunit polypeptide of AChE is the only polypeptide detectably solubilized by PIPLC. This selective removal of AChE does not affect the amount of acetylcholine released from intact synaptosomes upon K+ depolarization. PIPLC also quantitatively solubilizes AChE from the surface of intact bovine and rat erythrocytes, but only partially solubilizes AChE from human and mouse erythrocytes. The AChE released from rat and human erythrocytes by PIPLC migrates as a approximately 7S species on sucrose gradients, corresponding to a catalytic subunit dimer. PIPLC does not solubilize particulate AChE from any of the brain regions examined of four mammalian species. Several other phospholipases tested, including a nonspecific phospholipase C from Clostridium welchii, fail to solubilize AChE from Torpedo synaptic plasma membranes, rat erythrocytes, or rat striatum.     

Continuous hydrolysis of sucrose by invertase adsorbed in a tubular reactor

Studies were made of invertase adsorption on Amberlite ion exchange resins. Up to 4000 units of adsorbed enzymatic activity (aea) were obtainedper g of IRA 93 resin; for an aea of 1600 units, the maximum ratio of aea over units of soluble enzyme used for adsorption was close to 50%. Nodesorption occurred during extensive washing at 30°C with 0.01M sodiumacetate buffer at pH 5. Progressive desorption of aea from the invertase–IRA 93 complex occurred when buffer molarity and temperature were increased. Desorption differed only slightly when the buffer pH was 3 or 5. Theoptimum pH of aea was 3.2 with IRA 93 resin, and varied between 3.2 and 5.1with other resins, depending on their anionic or cationic nature. Batch hydrolysis of sucrose by IRA 93–adsorbed invertase followed 1st order kinetics with respect to the substrate concentration, as in the case of soluble invertase. Continuous sucrose hydrolysis with IRA 93–adsorbed invertase was performed in a tubular reactor, and the percent conversion was experimentally determined as a function of the flow rate. The reaction was experimentally determined 50% (w/v) sucrose solution, at pH4 and 30°C; at the selected flow rate, the ratio of sucrose hydrolysis remained constant and close to 76%. This shows that invertase was not desorbed from the tubular reactor. Some continuous hydrolyses were performed with an industrial sucrose solution: enzymatic activity seemed to be stable for anextended period for time (1 month) at 30°C and pH 3 or 4.     

Cholinergic neurons in the lamina ganglionaris of the blowfly Calliphora erythrocephala

Summary The distribution of putative cholinergic neurons in the lamina of the blowfly Calliphora erythrocephala was studied by immunocytochemical and histochemical methods. Three different antibodies directed against the AChsynthesizing enzyme, choline acetyltransferase (ChAT), revealed a cholinergic population of fibres running parallel to the laminar cartridges, which have branch-like structures at the distal lamina border. Cell bodies in the chiasma next to the lamina border were also labelled by the anti-ChAT antibodies. Monopolar cell bodies in the nuclear layer were faintly labelled. The distribution of the acetylcholine hydrolyzing enzyme, acetylcholine esterase (AChE), was revealed by histochemical staining and was similar to the ChAT immunocytochemistry. The arrangement of ChAT positive fibres in transverse and longitudinal sections and the distribution of AChE stained fibres indicate that the amacrine cells of the lamina are cholinergic cells.We dedicate this work to Prof. F. Zettler who passed away in fall 1988: K.-H. Datum, I. Rambold