Histochemical and cytochemical localization of acetylcholinesterase in the cerebellar cortex of the chicken

The localization of acetylcholinesterase (AChE) was studied in the cerebellar cortex of the crossbred trembler chickens by means of histo- and cytochemical methods. No essential differences between the crossbred normal and the crossbred trembler chickens were observed. The common results were as follows: Under a light microscope AChE activity was predominantly evident in the molecular layer, and secondly in the granular layer. AChE was ultrastructurally distributed principally in the cisternae of rough endoplasmic reticulum (ER) and in a part of nuclear envelope of the Purkinje, the Golgi and some of the basket and granule cells, and in a portion of the sacculus of the Golgi apparatus of the Purkinje cell only. In dendrites and the initial axon of the Purkinje cells the smooth ER also showed AChE activity. Although dendritic terminals of the Golgi cells contained AChE reaction products, the axon terminal did not. Some of the afferent terminal fibers forming the cerebellar glomerulus exhibited weakly a positive AChE reaction, while others in the vicinity did not show any AChE activity at all. However, the enzyme reaction product was localized in the intercellular spaces between a presynaptic afferent terminal and the postsynaptic granule cell dendritic terminals in the glomerulus. In addition, AChE activity was found in the form of spots in the intercellular spaces of both molecular and granular layers.     

Histochemically demonstrable esterase activity in the hypothalamus of the developing rat

Summary The distribution of the acetylcholinesterase, non-specific cholinesterase and non-specific esterase activity has been investigated histochemically in the hypothalamic neurons during the ontogenic development of the rat.Acetylcholinesterase activity is located in the supra-optic and para-ventricular nuclei mostly, but some activity is present in the other nuclei and in the median eminence of the adult rat, as well. The supra-chiasmatic neurons are always negative. The activity of non-specific cholinesterase was encountered in the endothelial cells of the capillaries, in the glia and in the ependymal cells especially around the supra-optic and para-ventricular neurons. The localization of the non-specific esterase was similar to that of the non-specific cholinesterase, but in addition activity is seen in the supra-optic and para-ventricular perikarya, in the parvo-cellular neurons of the tuberal area and in the median eminence. No sexual differences were seen in the distribution of the estrase activity.The appearance of acetylcholinesterase took place already before birth. At about the 16th post-coital day the area from which the arcuate and ventro-medial nuclei will differentiate was positive for acetylcholinesterase. A strong activity in these nuclei was observed during the critical period of the sexual differentiation of the rat hypothalamus (0–10 postnatal days). In the development of the non-specific cholinesterase and esterase no similar variation was seen. Acetylcholinesterase and non-specific esterase were seen in the neurosecretory nuclei before birth, non specific cholinesterase after birth, and non-specific esterase in the parvo-cellular neurons during the first post-natal week.Supported by a grant from The Finnish Medical Society Duodecim.     

Unequal patterns of development of succinate-dehydrogenase and acetylcholinesterase in Purkinje cell bodies and granule cells isolated in bulk from the cerebellar cortex of the immature rat

Abstract— –A preparative procedure for the isolation in bulk of two cellular populations of the cerebellar cortex of the immature rat, the granule cells and the Purkinje cell bodies, is described. The procedure is used to delineate the developmental pattern of succinate-INT-reduclase (EC 1.3.99.1) and acetylcholinesterase (EC 3.1.1.7) in the crucial period of cerebellar maturation, i.e. between 12 and 19 days postnatally. Although the overall yield of neuronal RNA diminished with age, the proportion of RNA in the Purkinje cell body fraction increased while that in the granule cells decreased and microscopic examination of the fractions confirmed this result. The yields of succinate-INT-reductase and of acetylcholinesterase in the fractions paralleled the yields of RNA. A significant finding was the trend toward diminishing specific activities (units/μg of RNA) with age of both enzymes in the Purkinje cell bodies as against the opposite, upward trend of their specific activities in the granule cells. An additional finding of interest was the different ratio of true acetylcholinesterase/total cholinesterase activity in the two cell types, with the granule cells consistently exhibiting higher true acetylcholinesterase values than the Purkinje cell bodies. The present report thus supplements the histoenzymological data on the developing rat cerebellum in that it reveals specific differences in the enzymatic development of two different cerebellar types, a finding which was greatly facilitated by the availability of the procedure for their bulk isolation.     

Acetylcholinesterase activity in developing skeletal muscle cells

Acetylcholinesterase activity has been demonstrated biochemically and cytochemically in developing chick embryo skeletal muscle cells growing in culture. The enzyme shows the same pattern of drug sensitivity as that of adult skeletal muscle acetylcholinesterase and in present in cultured myogenic cells before the time of cell fusion, the formation of myotubes, and the subsequent increase in rate of myosin synthesis. Myogenic cell fusion is accompanied, however, by a large increase in activity of acetylcholinesterase. The enzyme activity is restricted in these cultures to myogenic cells. Neighboring fibroblasts show no cytochemical responses when challenged with techniques showing intense activity in myoblasts and myotubes. In addition, evidence is presented which strongly suggests that acetylcholinesterase activity in dividing myogenic cells is not constant over the cell cycle.     

Disrupted cerebellar cortical development and progressive degeneration of Purkinje cells in SV40 T antigen transgenic mice.

SV40 T antigen (Tag) expression directed to cerebellar Purkinje cells resulted in the generation of three transgenic mouse lines that displayed ataxia, a neurological phenotype characteristic of cerebellar dysfunction. Onset of symptoms and cerebellar pathology, characterized by specific Purkinje cell degeneration, appeared to be directly dependent upon transgene copy number. The SV5 line (containing > 30 transgene copies), exhibited embryonic transgene expression that caused selective death of immature Purkinje cells and a subsequent block in cerebellar development and ataxia at 2 weeks. The developmental effect of the disruption of Purkinje cells in SV5 mice suggests that a normal complement of these cells is required for early development of the cerebellar cortex, especially granule cell proliferation and migration from external to internal layers. Transgene expression in a second line, SV4 (10 copies), was detectable during the second postnatal week. Death of mature Purkinje cells in the SV4 line resulted in onset of ataxia at 9 weeks. Ataxia in a third line, SV6 (2 copies), was detected after 15 weeks. The distinct cerebellar phenotypes of the SV4-6 lines correlate with specific Tag-induced Purkinje cell ablation as opposed to tumorigenesis.     

Comparative distribution of acid phosphatase and simple esterase in the mouse neocortex and hippocampal formation

The present study deals with the detailed distribution of acid phosphatase (AcP) and simple esterase (SE) in different layers of the neocortex and hippocampal formation of the mouse brain. The neurons, in general, had moderate to intense enzyme activity for AcP and mild to moderate activity for SE. The AcP activity dominated in the neuronal population as compared to the neuropil; the neuropil stained mildly for SE. The large pyramidal cells in the neocortex and cornu ammonis, and the granular cell layer of the gyrus dentatus, demonstrated strong enzyme activity both in AcP and SE preparations. The role of AcP and SE has been discussed in relation to various structures of the neocortex and hippocampal formation.     

Cre recombinase expression in cerebellar Purkinje cells

The cerebellar cortex and its sole output, the Purkinje cell, have been implicated in motor coordination, learning and cognitive functions. Therefore, the ability to generate Purkinje cell-specific mutations in physiologically relevant genes is of particular neurobiological interest. A suitable approach is the Cre/loxP strategy that allows temporally and spatially controlled gene inactivation. Here, we present the characterization of transgenic mouse strains expressing Cre recombinase controlled by the L7/pcp-2 gene. Endogenous L7/pcp-2 protein is expressed exclusively in Purkinje cells and retinal bipolar neurones. Recombination was detected by beta-galactosidase histochemistry in tissues from crosses of the L7/pcp-2:Cre transgenic lines with two different indicator strains, GtROSA26 and ACZL. Purkinje cells in all folia of the cerebellum displayed intense beta-galactosidase staining, whereas only few blue cells were observed in the retina and other parts of the CNS. Thus, these transgenic lines are potentially of great importance for genetic manipulations in cerebellar Purkinje cells.     

Cytochemical research on the matrix activity and status of the histone component of the neurocyte chromatin in the rat cerebellar cortex during postnatal differentiation

Evident differences in the ammoniacal silver staining pattern of histones were demonstrated for neurones of different layers of adult rat cerebellar cortex. These differences were formed during postnatal differentiation. It has been also shown for Purkinje and granular cells that time-course of age-dependent changes in histone staining are not coincident with that for template activity of these cells.     

Decapitation impacting effect of topically applied chlorpyrifos on acetylcholinesterase and general esterases in susceptible and resistant German cockroaches (Dictyoptera: Blattellidae)

The effect of topically applied chlorpyrifos on acetylcholinesterase and other esterases in heads and decapitated bodies of CSMA and Crawford German cockroaches was examined with spectrophotometric enzyme assay and native polyacrylamide gel electrophoresis. The toxicity of chlorpyrifos was greatly reduced in decapitated CSMA male cockroaches with LD50 value 17.1-fold higher than that of normal CSMA cockroaches. Acetylcholinesterase activity from heads was significantly higher in the Crawford compared with the CSMA strain and did not change until 24 h after chlorpyrifos in vivo treatment in both strains. The p-nitrophenyl butyrate (NPB) esterase activities from both heads and decapitated bodies of the resistant Crawford strain were significantly greater than the susceptible CSMA strain. The p-NPB esterase activity was significantly inhibited by chlorpyrifos in vivo treatment, and total p-NPB esterase activity was significantly reduced in decapitated bodies compared with heads of both strains. Native polyacrylamide gel electrophoresis (PAGE) analysis of extracts solubilized with Triton X-100 from heads and decapitated bodies revealed five major esterase bands and an acetylcholinesterase (AChE) band with a high capability of hydrolyzing alpha-naphthyl butyrate and acetylthiocholine, respectively. In the heads of susceptible CSMA male cockroaches, the activity of mobile isozymes d1 and d2 was completely inhibited at 24 h after chlorpyrifos application, and isozyme e was partially inhibited. In contrast, isozymes c1 and c2 from the decapitated bodies of CSMA cockroaches were mostly affected at 24 h after the topical application of chlorpyrifos. The activities of acetylcholinesterase and esterase isozymes a and b from the decapitated body remained uninhibited in both strains. Inhibition of isozymes d1 and d2 seems to be more important in chlorpyrifos intoxication than acetylcholinesterase.     

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents ³. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells ¹¹ are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period ⁴. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.     

Activity of the climbing fiber innervating various Purkinje cells

Low-amplitude potentials (10-130 microV) related to the action of a distant branch of the climbing fiber, which elicits complex spikes of the reference Purkinje cell were revealed by means of potential averaging synchronously with complex spikes of Purkinje cells in 10 out of 255 paired records of cerebellar Purkinje cells activity and extracellular field potentials at interelectrode distances of 200-1500 microns. These potential waves had a stable form in independent sets of data. In 3 out of 10 cases, the low-amplitude potentials included a slow (about 100 ms in duration) component. In one case, both test and reference electrodes recorded both simple and complex spikes of different Purkinje cells so that complex spikes of both cells were practically synchronous (conditional probability of complex spikes p = 0.97, onset time difference 0.54 ms). Thus for the first time in cerebellar physiology both simple and complex spikes activity of two Purkinje cells controlled by the same climbing fiber was recorded.     

多巴胺受体1在皖西白鹅小脑皮质发育中的表达

采用普通染色及免疫组化SABC染色法研究皖西白鹅小脑皮质的发育和多巴胺受体1(DRD1)阳性细胞在其发育中的表达.结果表明,小脑皮质在胚龄13 d(E13)由外向内分为外颗粒层(EGL)、浦肯野细胞层(PCL)和内颗粒层(IGL),E19由外向内分为EGL、分子层(ML)、PCL和IGL.随发育天数的增加,EGL的厚度和细胞层次呈先升后降的变化趋势,细胞密度逐渐下降;ML厚度逐渐增大,在E24到E28时增值最大;浦肯野细胞(PC)在E13、E19、E24和E28时随胚龄增大逐渐增大,在E28后趋于稳定,细胞密度随着发育天数的增加逐渐下降,在小脑皮质发育中还发现有一部分PC呈多层排列,且细胞层次逐渐变少;IGL厚度呈先升后降的变化趋势,细胞密度呈上升趋势.外颗粒层和内颗粒层在E13、E19、E24和E28时有DRD1阳性细胞表达,分子层在E24、E28、日龄7 d(P7)和15d(P15)有阳性细胞表达,PC在所检测的6个时段均有阳性表达.研究表明,小脑皮质的发育主要与细胞增殖、迁移和凋亡有关,外颗粒层的逐渐消失是以细胞迁移和凋亡为主,多层PC逐渐退化成单层是与细胞凋亡和正常突触联系的建立有关;DRD1在皖西白鹅小脑皮质发育中对外颗粒层细胞和PC起着重要作用.     

Histochemical distribution of acetylcholinesterase and simple esterases in the brain of squirrel monkey (Saimiri sciureus)

Summary The distribution of acetylcholinesterase (AChE) and simple esterases (SE) has been investigated in 15 thick fresh frozen sections of the squirrel monkey brain. Simple esterases are cellular enzymes, found in locations similar to those of acid phosphatase (AC), although not as abundantly. The neuropil in most of the nuclei of the brain shows stronger SE activity compared to the AChE reaction. The AChE activity is strong in nucleus caudatus and putamen, and appreciable quantities of this enzyme are found in nucleus fasciculus diagonalis band of Broca, habenular complex, nucleus interpeduncularis, cranial nerve nuclei, gray layers of colliculus superior, griseum pontis, nuclei olivaris inferior, cuneatus, gracilis, etc. The nucleus interpeduncularis has proved very interesting histochemically because it is rich in AChE and SE, as well as acid phosphatase, monoamine oxidase and lactic dehydrogenase. In the area postrema, the neurons give stronger SE activity than the parenchymal cells, while the AChE activity in both types of cells is similar. The molecular layer of cerebellum is stronger in AChE compared to SE, whereas the Purkinje cells and granule cells are strong in SE and show only negligible AChE activity. The lining cells of the choroid plexus, in addition to the ependymal cells, demonstrate a negligible to mild AChE reaction in comparison to moderately strong simple esterase activity. The significance of these observations has been discussed.This work has been carried out with the aid of Grant No. 00165 from the Animal Resources Branch, National Institute of Health and a grant (NGR-11-001-016) from The National Aeronautics and Space Administration. Thanks are due to Mrs. M. J. Nimnicht and Miss M. E. Rogero for their technical help.     

Pre and post synaptic NMDA effects targeting Purkinje cells in the mouse cerebellar cortex

N-methyl-D-aspartate (NMDA) receptors are associated with many forms of synaptic plasticity. Their expression level and subunit composition undergo developmental changes in several brain regions. In the mouse cerebellum, beside a developmental switch between NR2B and NR2A/C subunits in granule cells, functional postsynaptic NMDA receptors are seen in Purkinje cells of neonate and adult but not juvenile rat and mice. A presynaptic effect of NMDA on GABA release by cerebellar interneurons was identified recently. Nevertheless whereas NMDA receptor subunits are detected on parallel fiber terminals, a presynaptic effect of NMDA on spontaneous release of glutamate has not been demonstrated. Using mouse cerebellar cultures and patch-clamp recordings we show that NMDA facilitates glutamate release onto Purkinje cells in young cultures via a presynaptic mechanism, whereas NMDA activates extrasynaptic receptors in Purkinje cells recorded in old cultures. The presynaptic effect of NMDA on glutamate release is also observed in Purkinje cells recorded in acute slices prepared from juvenile but not from adult mice and requires a specific protocol of NMDA application.     

Muscarinic Stimulation Promotes Cultured Purkinje Cell Survival: A Role for Acetylcholine in Cerebellar Development?

Abstract: The survival and development of cerebellar neurons are under the control of interacting epigenetic signals. In the present study, we have examined interactive effects of nerve growth factor (NGF) and acetylcholine on in vitro cerebellar Purkinje cell survival. In initial experiments, dissociated rat cerebellar cultures were grown for 6–7 days in the presence of NGF and the stable cholinergic agonist carbachol. Simultaneous exposure to carbachol and NGF selectively increased Purkinje cell number, whereas neither agent was effective when tested alone. The increase in survival was blocked by the muscarinic antagonists atropine (0.1 µ M ) and pirenzepine (10 n M ), but not by methoctramine (25 n M ). Nicotine had no effect on survival when tested alone or in combination with NGF. The cerebellar cultures exhibited cholinergic neuronal traits: high-affinity choline uptake, and choline acetyltransferase and acetylcholinesterase activities. To determine whether transmitter produced in vitro triggers Purkinje responsiveness to NGF, cells were exposed to physostigmine, an acetylcholinesterase inhibitor. Physostigmine alone induced an atropine-sensitive increase in cell survival that was enhanced in the presence of NGF. These data suggest that the early expression of cholinergic traits plays a role in Purkinje development. Activation of muscarinic receptors triggers enhanced Purkinje survival in the presence of NGF.     

The effect of medium pH on rate of growth, neurite formation and acetylcholinesterase activity in mouse neuroblastoma cells in culture.

Cell division, neurite formation and acetylcholinesterase activity were examined in a clone (NBA2) of mouse neuroblastoma cells maintained for up to 120 hours in medium with pH values between 6.6 and 8.0. Growth rate decreased as pH was reduced from 7.8 to 6.6. Generation time at pH 7.4 was 25 hours, while the rate of cell division was negligible at pH 6.6. The total number of cells at stationary phase was less at the lower pH values. Neurite formation was enhanced markedly as the pH was reduced from 7.4 to 6.6. Acetylcholinesterase activity was 5- to 8-fold greater in cells exposed to medium at pH 6.6 than in cells maintained in medium at pH 7.4. The reduction in the rate of cell division and increases in neurite formation and acetylcholinesterase activity at pH 6.6 were reversible upon exposure of the cells to pH 7.4 medium. Cell viability was greater than 90% at all medium pH values over a period of 120 hours. Uncloned T-59 mouse neuroblastoma cells were affected similarly by changes in pH. These results show that manipulation of the environmental pH can reversibly alter growth, neurite formation, and acetylcholinesterase activity of mouse neuroblastoma cells in culture.     

The effect of SK channel modulators on the simple spike firing frequency in discharge of cerebellar Purkinje cells in laboratory mice

The activity of cerebellar Purkinje cells is studied as affected by CyPPA, a positive modulator of small-conductance calcium-activated potassium channels type 3 and 2 (SK3/SK2), and NS309, an activator of small- and intermediate-conductance calcium-activated potassium channels (IK/SK), in male two-month-old laboratory mice. CyPPA decreases the simple spike firing frequency in the discharge of Purkinje cells by an average of 25% 1 hour after application of 1mM of the compound. An application of 100 μM of NS309 reduces the simple spike firing frequency by an average of 47% during the same period. These results confirm the hypothesis that SK channels may be involved in the downregulation of simple spike firing frequency in Purkinje cells. The frequency-regulating effect of NS309 is stronger, suggesting that IK/SK channels play a decisive role in the regulation of Purkinje cell spiking activity. Since an increase of simple spike firing frequency in these cells is symptomatic of many locomotor activity disorders, e.g., spinocerebellar ataxia, the substances studied or their functional analogues might be of medicinal interest.     

Immunohistochemical characterization of the orphan nuclear receptor ROR alpha in the mouse nervous system.

ROR alpha is an orphan nuclear receptor. A deletion mutation in the ROR alpha gene leads to severe cerebellar defects, known as the staggerer mutant mouse. Although previous in situ hybridization (ISH) studies have shown that ROR alpha is highly expressed in the cerebellum, especially in Purkinje cells, and in the thalamus, sufficient immunohistochemical (IHC) study has not yet been presented. I demonstrate here the IHC analysis of ROR alpha using a specific anti-ROR alpha antibody, in adult and developing mouse nervous system. ROR alpha immunoreactivity was observed in the Purkinje cell and molecular layers of the cerebellum. The co-localization of ROR alpha with calbindin D(28K) (CaBP) and parvalbumin indicates that ROR alpha-positive cells were Purkinje cells, stellate cells, and basket cells. In addition to the cerebellum, strong to medium ROR alpha immunoreactivity was found in the thalamus, cerebral cortex (mainly in the layer IV), dorsal cochlear nucleus (DCN), suprachiasmatic nucleus (SCN), superior colliculus, spinal trigeminal nucleus, and retina. The immunostaining was restricted in nuclei of neurons. Developmentally, ROR alpha immunoreactivity was observed in the cerebellum and thalamus from embryonal day 16 (E16). The distribution of ROR alpha immunoreactivity and ROR alpha mRNA hybridization signal was almost coincident. However, the intensity of hybridization signal was not always parallel to that of immunoreactivity.     

Effect of thiamine and its derivatives on acetylcholinesterase activity of the blood and brain of albino mice

Thiamine, thiaminepyrophosphate and 4-methyl-5-beta-oxyethylthiazole are studied for their effect on the acetylcholinesterase activity in the brain, blood plasma and cells. The activity of acetylcholinesterase in blood cells is shown to be inhibited most of all by thiamine and thiazole. Acetylcholinesterase of the brain has been efficiently inhibited only by thiamine pyrophosphate.     

The cerebellar deficient folia (cdf) gene acts intrinsically in Purkinje cell migrations

Cerebellar deficient folia (cdf) is a recently identified mouse mutation causing ataxia and cerebellar abnormalities including lobulation defects and abnormal placement of a specific subset of Purkinje cells. To understand the etiology of the cerebellar defects in cdf mutant mice, we examined postnatal development of the cdf/cdf cerebellum. Our results demonstrate that Purkinje cell ectopia and foliation defects are apparent at birth, suggesting the cdf mutation disrupts the positioning of many, but not all, Purkinje cells during development. In addition to cerebellar abnormalities, we observed lamination defects in the hippocampus of cdf mutant mice, although neocortical defects were not seen. Furthermore, ectopic Purkinje cells in cdf/cdf mice express an increased level of Dab1 protein, as previously observed in mice with mutations in genes in the reelin signaling pathway. Lastly, analysis of cdf <-->ROSA26 chimeric mice demonstrated that the cdf mutation is intrinsic to Purkinje cells. We suggest that the cdf gene product is required in a subset of Purkinje cells, possibly to respond to Reelin signals.