Cholinesterase Staining Research Articles

Chronic myopathy induced by repeated bupivacaine injections

The effect of recurrent cycles of muscle fiber degeneration-regeneration was studied by repeated bupivacaine injections into the rat anterior tibial muscle. Injections of 0.6 ml of 0.75% bupivacaine were performed weekly for 6 months. The rats were allowed to recover for another 2 months and then killed. Histological and histochemical stains showed striking changes, including marked variability in fiber size, numerous internal nuclei, extensive fiber splitting and many whorled fibers. Combined staining for end-plate cholinesterase and terminal axons showed a markedly enlarged zone of terminal innervation. These findings suggest that the observed morphological changes usually attributed to a primary myopathic process may instead be the manifestations of impaired and incomplete regeneration occurring after cycles of degeneration-regeneration.

Vagotomy induced changes in acetyl cholinesterase staining and substance P-like immunoreactivity in the gustatory lobes of goldfish.

In teleost fish, the visceral sensory nuclei of the medulla are clearly separated into gustatory lobes and a general visceral sensory nucleus. Those branches of the vagus nerve which innervate the orobranchial cavity terminate in the vagal gustatory lobe, while the general visceral component of the vagus nerve terminates in the separate general visceral nucleus. In goldfish, the vagal lobe is a complex, laminated structure containing both motor and sensory elements. Transection of the vagus nerve results in distinct changes in the pattern of acetylcholinesterase staining and substance-P-like (SPL) immunoreactivity in the vagal lobe of goldfish. Following vagotomy, cholinesterase activity is eliminated from layers 4 and 6, both being layers in which primary gustatory afferent fibers terminate. In addition, SPL immunoreactive fibers disappear from the capsular root of the vagus nerve. These results indicate that the primary afferent input to the gustatory lobe involves at least two cytochemically distinct fiber types, one containing substance-P-immunoreactive material and the other containing or inducing acetylcholinesterase activity. Vagotomy also affects immunostaining and cholinesterase activity of the motoneurons deep in the vagal lobe. Following nerve transection, acetylcholinesterase activity is diminished, and SPL-immunoreactivity increased in the affected motoneurons. Similar changes were observed in axotomized motoneurons of other cranial nerve nuclei.

Muscle size and motor unit survival in mice.

The soleus muscles in neonatal and adult mice were surgically reduced in size on one side of the animal. The experimental and control muscles were excised 6-48 weeks later and the number of motor units in each muscle was estimated by stimulating the muscle nerve and counting step increments in the electromyogram recorded in vitro. Multiple innervation in individual muscle fibres was then assessed by intracellular recording and by visualization of end-plates in the light microscope with cholinesterase stain. Muscle fibres were counted in cross-sections of each muscle in the light microscope. Surgical reductions in the size of the muscle during the first 3 weeks of life produced correlated reductions in the number of motor units in the muscle. This could not be attributed to masking of motor units by multiple innervation, which was always less than 10% in these muscles. The loss of motor units was greatest following reduction in muscle size in newborn mice, whereas in 6-week-old mice there was no significant loss of motor units following the operation. Thus, survival of neonatal motor units shows an age-related dependence on the number of muscle fibres available for innervation. In control muscles there was a highly significant correlation between motor unit and muscle fibre numbers, which is consistent with the hypothesis that motor neurone survival during the embryonic period of cell death is dependent upon the number of muscle fibres available for innervation.

Medial septal projections to the dentate gyrus of the rat: electrophysiological analysis of distribution and plasticity.

Previous electrophysiological experiments in rabbits have suggested that medial septal stimulation activates dentate granule cells and evokes an associated negative field response at the granule cell layer, without an associated "dendritic" response. Anatomical studies have suggested that septal input to the granule cells may be to stratum moleculare, or close to the cell layer, or may not exist at all. The present experiments confirmed in rats anaesthetised with urethane that medial septal stimulation elicits single action potentials from cells in the granule layer. The associated negative field potential was maximal in the granule cell layer and there was no sign of a separate dendritic potential. The fibres responsible for this potential travel to the dorsal hippocampus in the fornix superior rather than the fimbria, taking the same course as the fibres which contribute to the dense cholinesterase staining just above the granule cell layer. Stimulation at 100 Hz for 1 s of either medial septal, or perforant path, input to the dentate granule cell layer produced long term potentiation of the subsequent evoked field responses to the stimulated pathway. The responses to the non-stimulated pathway were unchanged. Paired pulse stimulation produced both homosynaptic and heterosynaptic potentiation. These data suggest that medial septal input synapses close to granule cell bodies and produces a negative field potential which is a combination of dendritic and population spike potentials. Medial septal input also appeared to produce direct activation of hilar neurones, some of which may be basket cells or other interneurones. The data also show that long term potentiation is specific to this input, perhaps dependent on presynaptic mechanisms. Paired pulse potentiation, at least in the heterosynaptic case appears to depend on postsynaptic mechanisms.

Light and electron microscopic identification of nerve terminal sprouting and retraction in normal adult frog muscle.

A combined light and electron microscopic study was performed on neuromuscular junctions of normal adult frogs. In a previous investigation signs of new synapse formation, as well as abandoned former synaptic sites, have been observed in normal muscles (Wernig, Pécot-Dechavassine & Stöver, 1980a, b). Here we performed a detailed light and electron microscopic correlation to investigate those parts of junctions which, after staining for cholinesterase (ChE) and presynaptic axon terminals, were suspected either to be newly formed or sites abandoned by the presynaptic nerve and the Schwann cell. Thin presynaptic nerve branches, enclosed by Schwann cell sheaths along most of their length, formed synaptic contacts with the muscle fibre only at small circumscribed areas. In these regions post-synaptic secondary folds (invariably present at mature synapses) were either missing or were less well developed. At these small contacts, binding sites for fluorescein-labelled alpha-bungarotoxin were usually present. At other sites the ChE reaction product was present but an axon could not be detected in silver-stained preparations. Electron microscopic observation revealed post-synaptic secondary folds filled with ChE reaction product while the presynaptic axon and Schwann cell were missing. The sites with ChE remnants can thus be regarded as abandoned former synaptic contacts. No binding of fluorescein-labelled alpha-bungarotoxin could be detected at such sites. These findings confirm earlier suggestions that synaptic contacts in frog muscle are normally undergoing continual remodelling. The lack of binding sites for fluorescein-labelled alpha-bungarotoxin at abandoned synaptic sites suggests that a neural or Schwann cell factor is important for the maintainance of synaptic acetylcholine receptors.

Innervation and membrane specialization at neuromuscular junctions in submaxillaris muscle of the frog

The submaxillaris muscle of the frog after zinc iodide-osmium staining reveals the presence of polyneural innervation. Cholinesterase staining shows that the longer terminals have postsynaptic folds whereas the smaller terminals (up to 5 micron) lack them. Thin-section electron microscopy shows that muscle fibers with or without an M line have terminals with and without postsynaptic folds. The terminals with postsynaptic folds have presynaptic membrane outpocketings above folds. These outpocketings are rudimentary or absent in the terminals without postsynaptic folds. In longer junctions, the P face of the presynaptic membrane has double rows of paired particles on active zone ridges perpendicular to the axis of the muscle. In smaller junctions active zone ridges are rudimentary or absent and double rows of particles form various patterns. Postsynaptic active zones in longer junctions consist of clusters of particles leaving gaps in between, whereas in the smaller junctions they lack gaps. The polyneural innervation and different deployment of membrane particles at neuromuscluar junctions could be a factor responsible for different physiological properties of this muscle.

Endplate topography of denervated and disused rat neuromuscular junctions: Comparison by scanning and light microscopy

The effect of denervation and tetrodotoxin-induced muscle disuse on endplate structure was investigated in rat hind-limb muscles. The endplate was visualized by light microscopic cholinesterase staining and by scanning electron microscopy. Denervation resulted in a reduction in histochemically determined endplate dimensions proportionate to the decrease in muscle fiber circumference. Scanning electron microscopy, on the other hand, revealed a flattening or more often collapse of primary grooves with a reduction in the width of the endplate but no longitudinal shrinkage. Primary groove area per se was not measurable due to the loss of primary groove structural integrity. Thus, the apparent histochemical diminution of endplate length after denervation was artefactual, probably due to loss of cholinesterase activity and impeded access of substrate. In disuse, cholinesterase staining revealed a similar reduction in endplate girth with fiber atrophy but with a corresponding increase in endplate length. Scanning electron microscopy of disused muscle fibers confirmed these histochemical findings and the overall preservation of primary groove area. Disuse also resulted in an increase in the number of intrasynaptic primary groove branches as visualized by scanning electron microscopy. Finally, a specialized endplate “raised area”, prominent in soleus muscle, was greatly reduced after disuse but much less so after denervation. Thus, after denervation, primary groove structural integrity is lost and the shape of the endplate passively follows that dictated by circumferential loss of surface membrane. In disused muscle, presence of an intact axon preserves the structure and area but not the orientation of the primary grooves which are distorted by fiber atrophy. Disuse also strongly affects other endplate surface structures visualized by scanning electron microscopy

Localization of cholinesterase activity in the outer plexiform layer of the larval tiger salamander retina

Acetylcholinesterase activity, which may be indicative of cholinergic synapses, has been ultrastructurally localized in the outer plexiform layer of the larval tiger salamander retina using the cholinesterase staining method of Karnovsky and Roots. In the presence of the butyrylcholinesterase inhibitor, ethopropazine, precipitate is localized to discrete ‘stain laminae’ about 300 nm in their largest dimension and bounded by a 10 nm wide electron-lucent gap. Stain laminae are predominantly found adjacent to horizontal cell axon terminals which were identified by serial section analysis. Stain precipitate has not been found associated with horizontal cell dendrites and in only one case was a lamina found adjacent to a bipolar cell dendrite. Intracellular deposits of cholinesterase stain precipitate were also observed. In some cases, stain is found adjacent to synaptic ribbons in photoreceptor terminals. Likely artifacts appear to have been ruled out by control experiments in which retinas either were not treated with cholinesterase stain or were treated in the presence of the specific acetylcholinesterase inhibitor, BW284C51. Thus, the stain laminae probably result from specific, highly localized areas of acetylcholinesterase activity. The implications of the present study on current theories of cholinergic function in the vertebrate retina are discussed.

Development of prestriate visual projections in the monkey and human fetal cerebrum revealed by transient cholinesterase staining

Cholinesterase (ChE) staining was used to reveal the timing and pattern of development of afferents to the prestriate visual cortex (areas 18, 19, 20, and 21 of Brodmann) in a series of developing human and monkey fetal brains. This investigation was possible because the nucleus pulvinaris of the thalamus, the main source of subcortical projections to the prestriate cortex, displays positive reactivity after thiocholine incubation during the last three quarters of gestation, while neighboring thalamic nuclei that project to the adjacent neocortical areas are unstained. Staining of the pulvinar and its prestriate projections passes through six broad stages. Stage I begins in both species at the end of the first third of gestation. Positively stained fibers originate from the pulvinar and enter but do not extend beyond the hemispheric stalk. During stage II, pulvinar axons gradually invade the intermediate zone of the occipital lobe, and in stage III they reach the level of the subplate zone. In stage IV, which occurs around mid-gestation in both species, cholinesterase-positive fibers accumulate within the subplate zone subjacent to the developing prestriate cortex. During stage V, ChE-positive fibers penetrate the prospective prestriate cortex but do not yet form the alternating columnar pattern characteristic of pulvinar input to this area in the adults. Rather, ChE activity is concentrated in two continuous bands situated within prospective layers III-IV and VI; also a narrow band is visible in upper layer I. In stage V a clear histochemical border forms between prestriate and striate areas with ChE activity in prospective area 17 limited mostly to the superficial strata of layers I and II. This histochemical differentiation precedes the emergence of cytoarchitectonic landmarks. During stage VI, which begins in the last fifth of gestation in both species, the pulvinar become progressively less stainable and its projections can no longer be traced by ChE histochemistry.

Transient cholinesterase staining in the mediodorsal nucleus of the thalamus and its connections in the developing human and monkey brain.

The histochemical and morphological maturation of the mediodorsal nucleus (MD) and its connections were compared in human and rhesus monkey using acetylthiocholine iodide and Nissl methods. Histochemical analysis in fetuses, neonates, and adults of both primate species revealed that MD passes through three major stages of cholinesterase (ChE) reactivity. In Stage I (up to about 16 fetal weeks in man; 9 fetal weeks in monkey), ChE staining gradually increases in the MD nucleus and is intense in axons directed toward the frontal lobe through the internal and external capsules. In Stage II (about 16-28 fetal weeks in man; about 9-14 weeks in monkey), ChE staining in MD reaches peak intensity so that reaction product in the neurons and neuropil blackens the entire nucleus in both species. In favorable planes of section, ChE-positive fibers appear to connect MD and the basal forebrain both of which stain intensely. ChE-positive fibers can also be traced from the lateral margins of MD to the subplate zone beneath the developing frontal cortical plate where they continue to accumulate before later invading the cortex with heaviest concentration in presumptive layers 3 and 5. In Stage III (after 28 weeks of gestation to 6 postnatal months in man; from about 14 fetal weeks until 2 postnatal months in monkey), except for scattered positive cells, ChE staining gradually disappears in MD and the formerly dense laminar pattern in the cortex begins to lighten. The dramatic but transient increase in ChE staining in MD during fetal development as well as the sequentially related changes in its projections indicate that this early appearing enzyme may play a role in the development of the frontal lobe by influencing the differentiation of thalamoprefrontal connections.

The autonomic innervation of the vasa nervorum.

Using catecholamine fluorescence and histochemical cholinesterase staining combined with quantitative image analysis a direct autonomic innervation of arteries, arterioles, venules, veins and arterio-venous anastomoses within peripheral nerves was demonstrated in normal as well as in chemically sympathectomized rats. The adrenergic nerves carry varicosities and were more diffused than acetylcholinesterase-containing nerves; the arteries and the arterio -venous anastomoses were more richly innervated. The findings that acetylcholinesterase-positive nerve fibres were unalterated by chemical sympathectomy and were revealed by short incubation times are indicative of a true cholinergic, and probably parasympathetic, innervation of the vasa nervorum. The possible importance of the autonomic innervation of the vasa nervorum in the pathogenesis of some diseases of peripheral nerves is discussed.

Simultaneous ultrastructural visualization of acetylcholinesterase activity and tritiated norepinephrine uptake in renal nerves.

In this investigation we have combined the methods of ultrastructural demonstration of acetylcholinesterase activity with electron microscopic autoradiography for the demonstration of norepinephrine uptake. The results show electron-dense deposits indicative of acetylcholinesterase activity associated with perivascular axons overlaid by concentrations of silver grains representing exogenous tritiated norepinephrine. Forty-five percent of the intervaricose regions and 19% of the varicosities overlaid by autoradiographic grains showed "moderate" amounts of cholinesterase staining. A greater proportion of autoradiographic grains was observed on the varicosities than in the intervaricose regions; however, the amount of acetylcholinesterase activity was greater in the intervaricose regions than in the varicosities. This investigation provides evidence for the presence of periaxonal acetylcholinesterase staining in adrenergic axons in the rat kidney.

Neostigmine augments responses of the rat anococcygeus muscle to field stimulation.

1 The effects of neostigmine on noradrenergic transmission have been studied in the field stimulated, isolated anococcygeus muscle of the rat. 2 In those muscles where the excitatory response to field stimulation was not completely inhibited by guanethidine (5 X 10(-6) to 10(-5) M) or phentolamine (10(-6) M), atropine (5 X 10(-8) M) gave no further inhibition of the response. 3 The shape of the response to field stimulation was altered in a dose-dependent manner by neostigmine (5 X 10(-7) to 5 X 10(-6) M), such that a 'shoulder' appeared during the relaxation phase. The 'shoulder', present at all stimulation frequencies tested between 3 and 40 Hz, was abolished by atropine (5 X 10(-8) M) and unaffected by tubocurarine (10(-6) M). 4 Neostigmine (2.5 X 10(-6) M), whether alone or in the presence of atropine (5 X 10(-8) M), had no effect on the uptake or stimulation-induced release of [3H]-noradrenaline. 5 Using electron microscopy, small Schwann/axon bundles close to smooth muscle cells rarely showed cholinesterase staining, whereas larger bundles at the outer serosal aspects of the muscle exhibited dense staining. 6 It is concluded that the observed effects of neostigmine are not due to a presynaptic effect on noradrenergic transmission.

Development of muscles in the dorsal foreleg of rat embryos. A light microscopic study with the in situ cholinesterase staining technique.

The development of muscles from the dorsal side of the forelegs from 13- to 21-day-old rat embryos was investigated under a light microscope. The muscle blastemata and individual muscles were stained in situ with the cholinesterase technique. The first muscle blastemata are visible on the early day 13. It appears that mainly myotubes are stained. The antebrachial and brachial extensor muscles form separated anlagen which connect on the late day 13 in the proximal region of the extensor carpi ulnaris muscle. The individualization of the muscles in a muscle blastema takes place on days 13 and 14. On day 15 all extensor muscles are visible. However, at this time the inserting points of some of these muscles are not yet visible after staining with alcian blue. On the early day 16 the motor end-plates are conspicuous. Due to the content of unspecific cholinesterase in rat embryos the tendons are also stained on day 16. Muscles and tendons remain stainable until birth. In addition to the muscles also the nerves, especially the epifascial nerves, stain very well with the acetylcholinesterase reaction.

Constrictor actions of acetylcholine, 5-hydroxytryptamine and histamine on bovine coronary artery inner and outer muscle.

1. In bovine coronary arteries, cholinesterase staining showed an extensive cholinergic innervation at the adventitia-media junction, and some cholinesterase in the outer but not inner smooth muscle.2. Acetylcholine or methacholine caused large, atropine-sensitive contractions of outer muscle but caused little contraction of inner muscle.3. Fluorescence microscopy for monoamines and for histamine, supported by chemical assays, showed no adrenergic innervation but showed numerous fluorescent cells in the adventitia and the outer 50% of the media which stained as mast cells and contained large amounts of histamine and noradrenaline and some dopamine, but little 5-hydroxytryptamine (5-HT).4. 5-hydroxytryptamine (acting by D receptors) and histamine (acting by H(1) receptors) in high concentrations caused large contractions, of similar size, in inner and outer muscle. In given submaximal concentrations they generally caused more contraction of outer than inner muscle, particularly in the case of histamine, provided that imipramine or desipramine was present to inhibit uptake of the agents by mast cells which were present in the outer part of the artery wall.5. Without blockade of uptake, 5-HT applied to the arteries in submaximal concentrations caused less contraction of outer than inner muscle; histamine still caused significantly more contraction of outer than inner muscle.6. The findings indicate that the cholinergic constrictor nerves of these arteries, unlike adrenergic constrictor nerves of other systemic arteries, act almost solely on outer muscle of the vessel wall; and that mast cells give considerable protection against constriction by 5-HT, but little against histamine, reaching the vessel from its adventitial surface.

Morphometric studies on tenuissimus muscle spindles in the cat.

Muscle spindles were studied histochemically in serial transverse sections of 42 cat tenuissimus muscle specimens. Staining for myofibrillar adenosine triphosphatase was employed to identify nuclear bag 1, nuclear bag 2, and nuclear chain intrafusal muscle fibers. The nuclear chain fibers were further subdivided into three categories according to their polar length and the intensity of their staining for nicotinamide adenine dinucleotide tetrazolium reductase. A total of 430 spindle poles were surveyed. The mean spindle content of bag 1, bag 2, and chain fibers was established. The mean polar length of intrafusal fibers as well as that of the intracapsular and extracapsular spindle regions was determined. A cholinesterase (ChE) staining technique was used to demonstrate the termination sites of motor axons along intrafusal fibers. Two types of circumscribed ChE deposits. The "rim" and the "plate," occurred on the fibers. The nuclear chain fibers usually carried both the ChE rims and plates, while most nuclear fibers displayed only the plates. The ChE plates were assessed in term of their appearance, staining intensity, length, and location along the fibers. The mean number of ChE plates found along the fibers was established for each of the various intrafusal fiber types. These histochemical observations are discussed with regard to the current concepts of cat spindle morphology and motor innervation. The results suggest a degree of predictability in the spindle fiber content and in the distribution of motor nerve terminals along intrafusal muscle fibers, at least in the tenuissimus muscle.

The distribution of acetylcholinesterase and choline acetyl transferase in the cerebellum and posterior lateral line lobe of weakly electric fish (Gymnotidae)

Cholinesterase was demonstrated in the lobe of the cerebellum but not the corpus cerebelli of weakly electric gymnotid fish. It had a patchy distribution in the granule cell layer and was very dense in the molecular layer; the cholinesterase staining was also dense in the contiguous molecular layer of the subjacent electrosensory region. Choline acetyltransferase was also found in far greater amounts within the electrosensory region and caudal lobe of the cerebellum than within the corpus cerebelli itself.

A method for visualizing axons and endplates throughout whole mounts of skeletal muscles using combined silver and cholinesterase stain.

A silver-cholinesterase stain is described which allows the visualization of the complete axonal tree, perineurial sheaths, motor nerve terminals and the cholinesterase at neuromuscular junctions throughout whole mounts of relatively thin muscles. The method is rapid, reliable and simple and allows the preservation of topographical information regarding both terminal and nodal sprouting in the whole muscle.

An immunohistochemical study of enkephalins and other neuropeptides in the striatum of the cat with evidence that the opiate peptides are arranged to form mosaic patterns in register with the striosomal compartments visible by acetylcholinesterase staining

This report presents immunohistochemical evidence for a modular arrangement of enkephalin and substance P in the striatum of cats and kittens. In cross-sections from the adults, met-enkephalin immunoreactivity in the caudate nucleus was concentrated in discrete, variably shaped macroscopic patches (0.2–0.5 mm wide) spaced at roughly 0.7–1.0 mm intervals. Patches appeared only occasionally in the putamen but there were larger districts of high immunoreactivity in the nucleus accumbens. In sections incubated with antiserum to substance P, both patches of dense immunoreactivity and zones of low immunoreactivity were present in the caudate nucleus. Staining in the putamen was fairly homogeneous, while in the nucleus accumbens it was patterned and especially intense dorsally. A detailed comparison was made between the distribution of the enkephalin-rich patches and the circumscript zones of low acetylcholinesterase activity (striosomes) visible in serially-adjoining sections by the thiocholine method. There was a striking registration of the two sets of figures, nearly every cholinesterase-poor patch corresponding to an enkephalin-positive zone. Correspondences were more complicated in the substance P stain but many pale zones and some dark zones were aligned with the enkephalin-rich cholinesterase-poor figures. No comparable clumping of reaction product was visible in sections processed for somatostatin immunoreactivity. In the caudate nucleus of young kittens, small dark patches of enkephalin and substance P immunoreactivity also appeared. In the cholinesterase stain there were both light and dark patches, and it was mainly with the dark zones that the enkephalin-positive patches (and some substance P patches) were in register. We conclude that the caudate nucleus is divided up into pharmacologically distinct compartments, and that although these are present at birth, their final histochemical composition is established postnatally.

Localization of choline acetyltransferase at neuromuscular junctions.

Choline acetyltransferase (ChAc) was localized at the neuromuscular junctions of rabbit diaphragm with immunohistochemical staining, which produced a brown color, in combination with histochemical staining for cholinesterase, which produced a blue color, in the same section. The innervations of nerve terminals at the neuromuscular junctions were comparable when a silver impregnation was substituted for the immunohistochemical staining of ChAc.