Acetylcholinesterase-positive Neurons Research Articles

Interleukin-6 improves the survival of mesencephalic catecholaminergic and septal cholinergic neurons from postnatal, two-week-old rats in cultures

Interleukin-6 (human recombinant) supported the survival of cultured mesencephalic, catecholaminergic and septal cholinergic neurons from postnatal, two-week-old (P13-P15) rats. Significantly, more catecholaminergic neurons, stained by monoclonal anti-tyrosine hydroxylase antibody, were found in cultures supplemented with interleukin-6 at a concentration of 5 ng/ml than in cultures not treated with interleukin-6. The optimal dose used was 50 ng/ml. The survival effect of interleukin-6 on postnatal rat, tyrosine hydroxylase-positive neurons was observed both in cultures using serum-containing and serum-free medium. Contents of dopamine and noradrenaline in cultures with interleukin-6 were also larger than in control cultures. Interleukin-6 also increased the survival of cultured embryonic (E17) rat midbrain tyrosine hydroxylase-positive neurons. The effect on these neurons was, however, smaller, and the optimal dose of interleukin-6 was nearly 5 ng/ml. Interleukin-6 also supported the survival of cultured postnatal (P13) rat septal cholinergic neurons, visualized by acetylcholinesterase staining. The concomitant addition of mouse nerve growth factor (100 ng/ml) and interleukin-6 (50 ng/ml) had a synergetic effect on the survival of acetylcholinesterase-positive neurons in culture. Our data suggest that the survival of cultured tyrosine hydroxylase-positive, mesencephalic, and acetylcholinesterase-positive, septal neurons from postnatal two-week-old rats was supported by interleukin-6, just as there was a different dose dependency of interleukin-6 on the cultured postnatal neurons compared with embryonic neurons.

Influence of soluble environmental factors on the development of fetal brain acetylcholinesterase-positive neurons cultured in a chemically defined medium: comparison with the effects of l-triiodothyronine ( l-T 3)

In cerebral hemisphere neuronal cultures derived from 15-day-old rat embryos, the addition of l-triiodothyronine ( l-T 3) or nerve growth factor (NGF) enhanced the expression of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in a dose-dependent manner. When cultures were supplemented with both agents at maximal effective concentrations, the stimulation in ChAT and AChE activities was significantly greater than the sum of the individual effects. Conversely, when the cultures were exposed to astrocyte conditioned medium grown in the presence or absence of l-T 3 ( CM + l- T 3 or CM − l- T 3 ), laminin and fibroblast growth factor (FGF), ChAT and AChE activities were not stimulated above those of control cultures when added alone or in combination with l-T 3. Furthermore, l-T 3, NGF, CMs, laminin and FGF did not affect AChE + cell survival, but significantly increased neurite outgrowth and branching with NGF and l-T 3 being the most powerful agents followed by CMs, laminin and FGF. Additionally, the simultaneous addition of l-T 3 with either laminin or FGF in culture, caused an additive effect to the effect of l-T 3 in the neurite density of AChE + cells with both agents. This study shows that (1) thyroid hormones do not act through the regulation of soluble neurotrophic factors produced by astroglial cells, (2) thyroid hormones interact with the effect of NGF on ChAT and AChE activities, (3) the regulation of ChAT and AChE activities and the neurite outgrowth are independently regulated, and (4) the regulation of ChAT and AChE activities is very specific compared with that of neurite outgrowth. Thus, our recent immunocytochemical demonstration of l-T 3 nuclear receptors (TNR), in a subpopulation of AChE + cells correlated with l-T 3 morphological effects suggests that some effects of l-T 3 on the maturation of a subpopulation of AChE + cells may result from a direct effect of this hormone with its specific nuclear receptors.

Morphological Findings in Long-Term Pancreatic Tissue Transplants in the Anterior Eye Chamber of Rats

Morphological changes in 21-day-old embryonic and 2-day-old postnatal rat pancreatic tissue fragments transplanted into the anterior eye chamber of homologous animals for 74, 77, 180, and 534 days were investigated using light and electron microscopic, acetylcholinesterase enzyme, and immunohistochemical methods. The pancreatic acinar cells degenerated and were not observed at this stage of transplantation. The ductal system proliferated and partly differentiated into endocrine cells that subsequently formed many new islets of Langerhans as well. The structures of beta-, alpha-, delta-, and pancreatic polypeptide cells were found to be intact even 1.5 years after transplantation. In addition to this, the organization of these cells inside the islets is similar to that of normal pancreatic tissue. The transplanted tissue fragments were well vascularized with blood vessels and innervated also by serotonergic cells and acetylcholinesterase-positive neurons. It is concluded that pancreatic tissue fragments, with the exception of the acinar component, can grow and survive with intact structure in the anterior eye chamber of homologous rats for up to 1.5 years.

Effects of nucleus basalis lesions on cerebral cortical concentrations of corticotropin-releasing hormone (CRH)-like immunoreactivity in the rat

The present study was designed to determine the effects of partial cholinergic denervation on parietal cortical corticotropin-releasing hormone-like immunoreactivity (CRH-LI) in the rat at different ages. Young adult rats received either unilateral or bilateral ibotenic acid infusions into their nucleus basalis, destroying most of the acetylcholinesterase-positive neurons in that region. Parietal cortical levels of CRH-LI were assayed 2.5, 10, 14 and 19 months after placement of nucleus basalis lesions. Parietal CRH-LI was elevated at 10, 14 and 19 months in bilaterally lesioned animals, while unilateral lesions had no effect on CRH-LI.

Interleukin 3 as a trophic factor for central cholinergic neurons in vitro and in vivo

We have found that interleukin 3 (IL-3), a growth factor for hematopoietic cells, is a novel trophic factor for mouse and rat central cholinergic neurons. It enhanced neurite outgrowth and elevated choline acetyltransferase activity. The effect seems to be specific for cholinergic neurons, since somatostatin release and glutamic acid decarboxylase and 2′,3′-cyclic nucleotide 3′-phosphodiesterase activities were not significantly influenced by IL-3. In vivo, IL-3 was infused into the lateral ventricles of rats after unilateral axotomy of the septohippocampal pathways. Two weeks later, the IL-3-treated animals showed significant numbers of acetylcholinesterase-positive neurons remaining in the septa[ region.

Immunocytochemical localization of thyroid hormone nuclear receptors in cultured acetylcholinesterase-positive neurons: A correlation between the presence of thyroid hormone nuclear receptors and l-tri-iodothyronine morphological effects

A monoclonal antibody against the rat liver l-tri-iodothyronine nuclear receptor and acetylcholinesterase cytochemistry were used for the localization of thyroid hormone nuclear receptors in acetylcholinesterase-positive cell nuclei in fetal rat cerebral hemisphere neuronal cultures. After 3 days in vitro, the ratio of acetylcholinesterase-positive cells that were immunoreactive for the thyroid hormone nuclear receptor to those not stained for this receptor (74-26%, respectively) remains unchanged despite an increase in the number of acetylcholinesterase-positive cells with time (from day 3 to day 21) in culture. Furthermore, the addition of 3 × 10−8l-tri-iodothyronine in culture did not modify this ratio or have an effect on the number of acetylcholinesterase-positive cells, but significantly increased the neurite density in those acetylcholinesterase-positive cells that were immunoreactive for the thyroid hormone receptor. Conversely, no difference in the neurite densities of those acetylcholinesterase-positive cells not stained for this receptor was observed when cultured in the presence or absence of thyroid hormone. In other experiments with the same fetal brain cultures, treatment of cultures for 8 days with l-tri-iodothyronine, beginning on culture day 20, demonstrated the presence of a critical period which occurs in vitro around day 20, since the stimulatory effect of l-tri-iodothyronine on immunoreactive acetylcholinesterase-positive cell neurite density is lost after 20 days in vitro.These results demonstrate, for the first time, the presence of l-tri-iodothyronine nuclear receptors in fetal rat acetylcholinesterase-positive neurons and the existence of a cellular heterogeneity in the distribution of the thyroid hormone receptor. The presence of these receptors in fetal brain acetylcholinesterase-positive neurons suggests that some effects of l-tri-iodothyronine on the maturation of a subpopulation of acetylcholinesterase-positive neurons may result from a direct effect of this hormone through an interaction with its specific nuclear receptors.

Localization of acetylcholinesterase positive neurons and substance P and enkephalin positive fibers by histochemistry and immunohistochemistry in the sympathetic intermediate zone of the developing human spinal cord

Localization of acetylcholinesterase positive neurons and substance P and enkephalin fibers were studied by histochemistry and immunohistochemistry in the intermediate sympathetic zone of the spinal cords of 39 human embryos/fetuses from gestation ages five to 40 weeks. Acetylcholinesterase positive neurons were observed in the nucleus intermediolateralis pars principalis as early as the fifth week of gestation. By the ninth to 13th weeks of gestation, positive neurons were also seen in the nuclei intermedialis pars funicularis, intercalatus spinalis and intercalatus pars paraependymalis. Increase in amount of these positive acetylcholinesterase neurons was demonstrated till term. Substance P and enkephalin fibers were initially observed by the eighth gestation week in the intermediolaterlis pars principalis nucleus and positive fibers were then detected in the nucleus intermedialis pars funicularis as well as the nucleus intercalatus spinalis by the 14th week of gestation. By the 26th week of gestation, all the major nuclei intermediolateralis par principalis, intermedialis pars funicularis, intercalatus spinalis and intercalatus pars paraependymalis has substance P and enkephalin fibers. Initial demonstration of acetylcholinesterase positive neurons appeared to be at an earlier stage than that of our substance P and enkephalin positive fibers.

Continuous presence of nerve growth factor is required for maintenance of cholinergic septal neurons in organotypic slice cultures

In co-cultures of rat septum and hippocampus, cholinergic neurons, identified by immunocyto-chemical techniques using antibodies against choline acetyltransferase, were found to be exclusively located in septal tissue. The presence of nerve growth factor during the entire growth period of four weeks increased the activities of acetylcholinesterase and choline acetyltransferase about 10-fold and strongly increased the number of acetylcholinesterase-positive neurons. Application of nerve growth factor yielded different effects depending on the age of the cultures. During the first two weeks in vitro, nerve growth factor enhanced the number of acetylcholinesterase-positive neurons, an effect which was no longer observed following later applications of nerve growth factor. Nerve growth factor increased the activities of cholinergic enzymes during all phases of in vitro development, but the effects of one-week applications were always considerably smaller than those observed following continuous application of nerve growth factor. The results of different application schedules suggest that the continuous presence of nerve growth factor is needed for maximal increases in cholinergic enzyme activities and maintenance of cholinergic neurons in septohippocampal co-cultures.

Neurons controlling the gill vasculature in five species of teleosts

A plexus of nerve fibers encompassing neuronal perikarya is present within the gill filament; it surrounds the proximal portion of the efferent filament artery and the efferent lamellar arterioles. This innervation resembles the pattern described for the area around the sphincter of the efferent filament artery: acetylcholinesterase-positive neurons and fibers, fast-fading yellow-fluorescent neurons and fibers, long-lasting green-fluorescent fibers. In addition, synaptic contacts between the different components suggest functional interrelationships. Nerves evidently control the efferent limb of the filament circulation including the sphincter of the efferent filament arteries, the proximal portion of the efferent filament arteries proper, and their corresponding efferent lamellar arterioles. However, the distal portion of this system is poorly innervated.

Degenerative Changes in Forebrain Cholinergic Nuclei Correlate with Cognitive Impairments in Aged Rats.

Degenerative changes in the forebrain cholinergic nuclei have been studied morphometrically in behaviourally characterized aged female Sprague-Dawley rats. In all regions analysed (medial septum, diagonal band of Broca, nucleus basalis, and striatum) the acetylcholinesterase-positive neurons were reduced in both size and number in the aged (24-months-old) rats as compared to the young (3-months-old) controls. The overall reduction in cell size amounted to between 20 and 30% and the overall reduction in cell number to between 27 and 45%. Impairment in learning and/or memory performance in the aged rats, as assessed in the Morris' water-maze task, was significantly correlated with both cholinergic cell size and cell number in the medial septum, and with cholinergic cell number in the diagonal band of Broca and in the striatum. In the nucleus basalis there was a trend in the same direction but it did not reach significance. In contrast to these degenerative changes in the cell body regions, no significant differences in cortical or hippocampal choline acetyltransferase activity were detected biochemically between the young and the aged rats, and the enzyme activity levels did not correlate with the degree of behavioural impairment in the aged rats. The present results provide evidence that all major forebrain cholinergic cell groups undergo degenerative changes with age in the rat, and that the most severe changes are found in those rats which display the most profound spatial learning impairments. Despite the severe changes at the cell body level, however, the choline acetyltransferase activity in the cortical projection areas are affected only to a minor degree, perhaps as a result of functional compensatory changes at the terminal level.

Neuronal degeneration in the pineal ganglion during the post-hatching development of the domestic fowl.

The frequency of pineal ganglia associated with the pineal tract, and the numbers of acetylcholinesterase-positive neurons in these ganglia were studied in the domestic fowl during the post-hatching period by means of the acetylcholinesterase method. Furthermore, the degeneration of nerve cells in pineal ganglia of 40-day-old domestic fowl was investigated in detail at the electron-microscopic level. The rate of pineal organs containing one or more ganglia was 50% in 2- to 13-day-old, 38% in 40-day-old, and only 10% in 1-year-old domestic fowl. In parallel, the number of acetylcholinesterase-reactive nerve cells that constitute individual pineal ganglia decreased after hatching. Various degrees of neuronal degeneration were found in the pineal ganglia: swelling of the endoplasmic reticulum, electron-dense degeneration of the cytoplasm, and pyknosis of the nerve cell nucleus. Clusters of macrophages containing numerous lysosomes filled with debris-like material were scattered in the ganglion. In addition, plasma cells were observed in association with degenerating nerve cells. These results confirm the suggestion that the loss of acetylcholinesterase-positive nerve cells in the pineal ganglia of the domestic fowl is due to naturally occurring, programmed neuronal cell death. This process is discussed with reference to phenomena of cell death observed in other components of central nervous system.

Influence of triiodothyronine ( l-T3) on the morphological and biochemical development of fetal brain acetylcholinesterase-positive neurons cultured in a chemically defined medium

In cerebral hemisphere cultures initiated from 15-day-old rat embryos, the number of acetylcholinesterase-positive (AChE+) cells increased from 6.8 ± 1.6 cells/well on day 3 to 112 ± 16 cells/well on day 15. With time in culture, AChE+ cells increased both in size of the perikarya and neurite length. The addition of l-triiodothyronine ( l-T3) at a concentration of 3 × 10 −8 M at the initiation of the culture had no effect on the number of AChE+ cells but significantly increased the size and neurite length of AChE+ neurons after 5 days in vitro. These morphological effects are associated with biochemical effects. l-T3 increased AChE activity in both a dose- and time-dependent manner (the stimulatory effect of l-T3 becomes significant between day 8 and day 15). Since a major part of AChE+ cells may be cholinergic neurons, we have also measured the effect of l-T3 on ChAT activity. l-T3 also increased ChAT activity in a dose and time dependent manner. Furthermore, treatment of cultures with l-T3 at different times in culture demonstrated the presence of a critical period which occurs in vitro around day 20, since the stimulatory effect of l-T3 on ChAT activity is lost after 20 days in vitro. Studies of the time necessary for l-T3 to increase both ChAT and AChE activities show that 2 days and 15 days, respectively, are required for l-T3 to significantly stimulate both enzyme activities. This in vitro analysis demonstrated the morphological effect of l-T3 on the size and the neurite length of AChE+ cells. These effects are associated with biochemical effects on ChAT and AChE activities. Thus, it appears that thyroid hormones regulate several steps of neuronal maturation.

Nerve growth factor promotes survival of cultured magnocellular cholinergic neurons from nucleus basalis of Meynert in postnatal rats

One of the possible causes of Alzheimer's disease is thought to be a lack of nerve growth factor (NGF), which plays an important role in the neuronal differentiation and cell survival of basal forebrain cholinergic neurons. In the present study, we report for the first time a direct in vitro effect of NGF on the survival of magnocellular cholinergic neurons of the nucleus basalis of Meynert. A dissociated culture of cholinergic neurons was prepared after dissecting out the nucleus from vibratome slices of postnatal rat forebrain. After culturing the neurons of this nucleus from 2-week-old rats for 6 days, the cell number of viable acetylcholinesterase-positive cholinergic neurons in the presence of NGF was found to be greater than that in the absence of NGF. Also, more extensive and denser acetylcholinesterase-positive neurites were observed in the NGF-treated culture. Higher activities of choline acetyltransferase were also observed in the NGF-treated culture. Cellular choline acetyltransferase activity, which was calculated by dividing the enzyme activity by the viable number of acetylcholinesterase-positive neurons for each well, was almost the same with or without NGF. These results mean that NGF enhanced cholinergic neuronal survival and cholinergic neurite regeneration, but did not induce cellular choline acetyltransferase activities.

Morphometric analysis of acetylcholinesterase-positive neurons in spinal cord organotypic roller tube cultures: [formula omitted] and [formula omitted], Department of Neurology, Harvard Medical School, The Arnold Center, New England Deaconess Hospital Boston, MA 02215, U.S.A., and Dana Research Institute, Beth Israel Hospital, Boston, MA 02215, U.S.A.

Morphometric analysis of acetylcholinesterase-positive neurons in spinal cord organotypic roller tube cultures: [formula omitted] and [formula omitted], Department of Neurology, Harvard Medical School, The Arnold Center, New England Deaconess Hospital Boston, MA 02215, U.S.A., and Dana Research Institute, Beth Israel Hospital, Boston, MA 02215, U.S.A.

Morphological analysis of acetylcholinesterase-positive neurons of the rat brain in culture and adult [formula omitted

Morphological analysis of acetylcholinesterase-positive neurons of the rat brain in culture and adult [formula omitted

Septal cholinergic neurons from postnatal rat can survive in the dissociate culture conditions in the presence of nerve growth factor

The survival effect by nerve growth factor (NGF) on the cholinergic neurons of postnatal rat septal neurons in culture was examined. When the septal neurons from 10 to 12-day-old rats were cultured without NGF, the activities of choline acetyltransferase gradually decreased during the period of cultivation. The addition of NGF to the culture prevented the decline of activities. And, the number of acetylcholinesterase-positive neurons in culture with NGF was found to be more than that without NGF, after 5 days in culture. These results suggest that NGF promotes the survival of septal cholinergic neurons from postnatal rats in culture.

Quantitative autoradiography of dopamine D 2 sites in rat caudate-putamen: Localization to intrinsic neurons and not to neocortical afferents

Dopamine D 2 receptors, labeled with [ 3H]spiroperidol or [ 3H]sulpiride, show a lateral-to-medial gradient in the caudate-putamen, with a more than two-fold greater density laterally than medially. It has been thought that D 2 receptors are located on at least two neuronal elements of the caudate-putamen, neurons intrinsic to this structure and axons whose cell bodies reside in the cortex. As a first step in establishing what neuronal elements underlie this heterogeneous organization of D 2 receptors, we took advantage of quantitative autoradiography to examine the association of these receptors with those elements. The present findings show that the D 2 sites are almost exclusively located on neurons whose somata reside in the caudate-putamen and are not located on terminals of corticostriatal axons. A detailed comparison of the distribution of histochemically identified acetylcholinesterase neurons with that of D 2 receptors in serially adjoining sections suggests a common organizational pattern. The density of [ 3H]spiroperidol sites in rat caudate-putamen was determined after unilateral injection of the neurotoxin quinolinic acid into this structure or after ablation of neocortical regions. Quantification of the tissue damage was achieved by acetylcholinesterase histochemistry (following diisopropylfluoro-phosphate treatment), as well as by thionin and luxol fast staining of sections adjacent to those used for [ 3H]spiroperidol autoradiography. In identically treated animals, biochemical determination of the extent of tissue damage was made utilizing assays for high-affinity [ 3H]choline and [ 3H]glutamate uptake in the caudate-putamen. In quinolinic acid-injected rats, the density of D 2 sites was decreased by 90–95% at the site of complete loss of large acetylcholinesterase-positive neurons. Other animals, given ablations of specific neocortical fields (medial prefrontal, motor, somatosensory) or of the entire parietal-frontal cortex of one hemisphere, showed no loss of caudate-putamen D 2 sites unless the cortical ablation caused accompanying damage of the caudate-putamen. In the caudate-putamen of all animals there was a close correspondence between the D 2 sites and the striatal neurons (and processes) that show strong acetylcholinesterase reactivity. We suggest that the caudate-putamen topography of D 2 sites is based largely on the internal organization of this structure and may preferentially involve acetylcholine-containing intrinsic neurons.

Selective kainic acid lesions in cultured explants of rat hippocampus.

The influence of the excitotoxin kainic acid (KA) on cultivated explants of rat hippocampus was investigated. Addition of 3 microM KA to the culture medium over 24-48 h induced a destruction of the pyramidal cells in the CA3 region, whereas the CA1 pyramidal cells and the granule cells were left undamaged. Higher concentrations (10-100 microM) of KA destroyed also the latter cell groups. The selectivity of the KA lesion at 3 microM was further indicated by the fact that the acetylcholinesterase-positive neurons in the hippocampus were not destroyed through KA administration and that the stereoisomer dihydrokainic acid was ineffective in inducing lesions. Application of tetrodotoxin did not protect the CA3 pyramidal cells from KA lesion, whereas gamma-glutamylaminomethylsulphonic acid (GAMS) only offered a very small, statistically not significant, protection. Baclofen protected the cultures slightly from KA lesions but not when added together with GAMS. Possible mechanisms responsible for the KA lesions in these cultures are discussed.

Neurotoxicity of monosodium-L-glutamate in pregnant and fetal rats.

Monosodium-L-glutamate given subcutaneously to pregnant rats caused acute necrosis of the acetylcholinesterase-positive neurons in the area postrema. The same effect has been observed in the area postrema of fetal rats. The process of neuronal cell death and the elimination of debris by microglia cells proved to be similar in pregnant animals and in their fetuses. However, embryonal neurons were more sensitive to glutamate as judged by the rapidity of the process and the dose-response relationship. These observations raise the possibility of transplacental poisoning in human fetuses after the consumption of glutamate-rich food by the mother.

Morphological evidence for the existence of multiple neuronal classes in the cat lateral superior olivary nucleus

This study characterizes morphologically the neurons residing within the matrix of the cat lateral superior olive (LSO), excluding the hili and myelinated axon envelope. Several light microscopic techniques including Golgi impregnations, Nissl stains, and acetylcholinesterase histochemistry were used, as well as electron microscopy. Five distinct classes of neurons have been identified: principal neurons, multiplanar neurons, marginal neurons, small neurons, and class 5 neurons. These neuronal classes differ in regard to their size and shape, dendritic organization, perikaryal synaptic density, and their relative numbers. Principal neurons compose approximately three-quarters of the LSO neurons. They are multipolar and uniplanar in their dendritic arborization, radiating from the hili in rostrocaudal planes perpendicular to the curvatures of the LSO. In transverse sections the principal cell perikarya are fusiform and bipolar, with mean dimensions of 23 X 11 microns. More than 60% of the surface of these cells is contacted by synaptic terminals. Multiplanar neurons (averaging 23 X 19 microns) compose only 11% of the LSO neuronal population. Their dendritic arborization is not restricted to any particular plane, and their somal surface receives synaptic contacts similar, in number and type, to principal cells. Marginal neurons, although they are similar to principal neurons in shape and dendritic arborization, differ in that they are generally smaller (averaging 20 X 10.5 microns). They also possess fewer axosomatic synaptic contacts (approximately 33%), are oriented perpendicularly to principal neurons, are limited in distribution to the contours of the LSO immediately beneath the myelinated axon envelope, and constitute only 4% of the neuronal population. Small neurons (mean dimensions = 9 X 8 micron) compose 8% of the LSO neurons. They possess a multiplanar array of primary dendrites and have nuclei with multiple deep infoldings. Small neurons have the fewest axosomatic synaptic contacts of all classes of LSO neurons (approximately 10%). Additionally, there are neurons that are similar to principal neurons, but receive fewer axosomatic contacts (approximately 33%). These cells have been tentatively identified as class 5 neurons until more information on this type allows for the assignment of a more descriptive name. A number of acetylcholinesterase-positive neurons are also found within the LSO, whose relationship to the other classes of neurons is presently unresolved. Possible functions of the multiple neuronal types are discussed.