TOPOGRAPHICAL AND SUBCELLULAR LOCALIZATION OF CHOLINE ACETYLTRANSFERASE IN RAT HIPPOCAMPAL REGION

—Choline acetyltransferase (ChAc) was localized in discrete layers in hippocampus regio superior and in area dentata. The highest activity in hippocampus was found in a narrow infrapyramidal zone, but high activities were also observed in the rest of stratum oriens and in stratum pyramidale. In area dentata the highest activities were found in a narrow supragranular zone and in hilus fasciae dentatae. The localization corresponded very closely to that of acetylcholinesterase. The main part of ChAc activity was confined to the synaptosome fraction. The results are compatible with the view that pyramidal and granular cells are excited by cholinergic boutons, mainly located on the basal or apical dendrites near the somata.     

THE DISTRIBUTION OF GLYCINE, GABA, GLUTAMATE AND ASPARTATE IN RABBIT SPINAL CORD, CEREBELLUM AND HIPPOCAMPUS

The distribution of glycine, GABA, glutamate and aspartate was measured among about 60 subdivisions of rabbit spinal cord, and among the discrete layers of cerebellum, hippocampus and area dentata. A more detailed mapping for GABA was made within the tip of the dorsal horn of the spinal cord. Spinal ventral horn and dorsal root ganglion cell bodies were analyzed for the amino acids and for total lipid. The distribution of lipid and lipid-free dry weight per unit volume was also determined in spinal cord. Calculated on the basis of tissue water, glycine in the cord is highest in lateral and ventral white matter immediately adjacent to the ventral grey. The distribution of GABA is almost the inverse of that of glycine with highest level in the tip of dorsal horn. It is most highly concentrated in the central 75% of Rexed layers III and IV. Aspartate in the tip of ventral horn is 4-fold higher than in the tip of the dorsal horn and 3 times the average concentration in brain. Glutamate was much more evenly distributed and is relatively low in concentration with slightly higher levels in dorsal than in ventral grey matter. Large cell bodies in both ventral horn and dorsal root ganglion contained high levels of glycine. As reported by others, GABA was found to be high in cerebellar grey layers, area dentata, and regio inferior of hippocampus. Glycine was moderately high in cerebellar layers but moderate to low in hippocampus and area dentata.     

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

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

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

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

Differential neuronal loss following early postnatal alcohol exposure

Neonatal rats were exposed to 6.6 g/kg of alcohol each day between postnatal days 4 and 10 while artificial-rearing procedures were used, in a manner which produced high peak and low trough blood alcohol concentrations each day. Gastrostomy controls were reared artificially with maltose/dextrin isocalorically substituted for alcohol in the milk formula, and suckle controls were reared normally by dams. The pups were sacrificed on day 10 and tissue sections (2 microns thick) were obtained in the sagittal plane through the cerebellum and in the horizontal plane through the hippocampal formation. Overall area measures were obtained for the hippocampus proper, area dentata, and cerebellum, along with areas of the cell layers of these regions. In the hippocampal formation, cell counts were made of the pyramidal cells of the hippocampus proper, the multiple cell types of the hilus, and the granule cells of the area dentata. In the cerebellum, cell counts of Purkinje cells, granule cells of the granular layer, granule cells of the external granular layer, and mitotic cells of the external granular layer were obtained from lobules I, V, VII, VIII, and IX. Alcohol selectively reduced areas and neuronal numbers in the cerebellum but had no significant effects on neuronal numbers in the hippocampal formation. Purkinje cells exhibited the greatest percent reductions, and cerebellar granule cells were significantly reduced in the granular layer but not in the external granular layer. All lobules showed these effects, but lobule I was significantly more affected than the other four lobules that were analyzed. The results demonstrate the differential vulnerability of selected neuronal populations to the developmental toxicity of alcohol exposure during the brain growth spurt.     

Distributions of choline acetyltransferase and acetylcholinesterase activities in layers of rat superior colliculus

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were assayed in samples dissected from sagittal sections through rat superior colliculus. The magnitude of ChAT activity was about half to equal that found in rat whole brain in all layers except stratum griseum intermediale, where the average activity was higher than whole brain. AChE activity was three to four times that found in rat whole brain in superficial layers and about the same as average brain in deeper layers, except in the statum griseum intermediale, where the average activity was about twice whole brain. Rostral-caudal gradients in both ChAT and AChE activities occurred in stratum griseum intermediale, with activities in the caudal region of some animals as high as four times those in the rostral. ChAT activity in samples associated with locations of patches or spots of AChE staining product in stratum griseum intermediale was significantly higher than in samples from "nonpatch" regions. Results are discussed relative to inputs into the colliculus, whose terminations may correlate in location with the distributions of the enzyme activities.     

QUANTITATIVE HISTOCHEMISTRY OF GLUTAMATE DECARBOXYLASE IN THE RAT HIPPOCAMPAL REGION

Abstract— The distribution of glutamate decarboxylase (GAD) was studied in hippocampus regio superior and in area dentata. In both regions the enzyme showed a bimodal distribution profile with peak activities in the molecular and pyramidal layers of hippocampus, and in the molecular and granular layers of area dentata. The pyramidal and granular layers contain the terminals and perikarya of inhibitory intrinsic neurones, the basket cells. The molecular layers contain neurones of similar morphology but with unknown function. GAD showed a very low activity in fimbria, a major pathway of fibres efferent and afferent to the hippocampal region.
The data are consistent with GAD being concentrated within the inhibitory local neurones of the hippocampal region.     

Quantitative succinate dehydrogenase histochemistry in the hippocampus of aged rats

Quantitative histochemical methods (microphotometric kinetic and end-point measurements, and morphometric analyses of reactive areas) were used to investigate the levels of succinate dehydrogenase (SDH) in the hippocampus of young adult (3-6 months old) and aged male rats (24-27 months old). Methodological studies concerning the demonstration of SDH activity, which were performed using hippocampi of young animals, revealed a linear relationship between the reaction time and the amount of reaction product for up to 20 min; kinetic (continuous) and end-point measurements provided the same results. In a number of experiments, it was established that an incubation medium consisting of 100 mM succinate, 10 mM sodium azide, 3 mM nitro blue tetrazolium chloride, 0.25 mM phenazine methosulfate, and 7.5% polyvinylalcohol in 0.05 M Hepes buffer (final pH 7.5) was optimal for quantitative SDH histochemistry in the hippocampus. Comparative quantitative investigations of SDH activity in rat hippocampi showed that, in most regions and layers of the hippocampus of both young and aged rats, the levels of SDH activity increased along the rostrocaudal axis of the hippocampus, i.e., higher levels were present in the caudal than in the rostral pole. In both groups, the highest SDH levels were observed in the molecular layer of the cornu ammonis (CA)-1, the CA-3, and the fascia dentata (middle and outer thirds), most of which are termination fields of the excitatory perforant path arising from the regio ento-rhinalis. Furthermore, in almost all of the investigated layers, the older animals exhibited lower SDH levels than young animals. These differences were statistically significant in the molecular layer of the fascia dentata and in most layers of the CA-3. The lower SDH levels in aged animals are discussed in relation to the reduced capacity for energy metabolism in the aging brain.     

Brain Regional Distribution of Glutamic Acid Decarboxylase, Choline Acetyltransferase, and Acetylcholinesterase in the Rat: Effects of Chronic Manganese Chloride Administration after Two Years

Abstract: Rats were treated chronically with manganese chloride from conception onward for a period of over 2 years in order to study the effects of manganese and aging on the activities of glutamic acid decarboxylase (GAD), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE) in hypothalamus, cerebellum, pons and medulla, striatum, midbrain, and cerebral cortex (which included the hippocampus). Manganese-treated 2-month-old and 24- to 28-month-old rats and age-matched controls were studied. In control rats during aging the activities of GAD decreased in hypothalamus (19%), pons and medulla (28%), and midbrain (22%) whereas the activities of AChE decreased in all regions (20–48%), particularly in the striatum (44–48%). Changes in ChAT activities in aging were observed only in one region—a decrease (23%) in the striatum. Life-long treatment with manganese appeared to abolish partially the decreases in aging in AChE activities in hypothalamus, cerebellum and striatum, and striatal ChAT activity. Manganese treatment also seemed to abolish the age-related decreases in GAD activities, since GAD activities in various brain regions of manganese-treated senescent rats were not significantly different from those of control young rats. These results are discussed in relation to other metabolic changes associated with aging and manganese toxicity.     

Acetylcholinesterase and butyrylcholinesterase activities in brain and plasma of freshwater teleosts: cross-species and cross-family differences

Brain and plasma acetylcholinesterase (AChE; EC 3.1.1.7) and plasma butyrylcholinesterase (BChE; EC 3.1.1.8) specific activities were assayed in 16 freshwater teleosts belonging to four families: Cyprinidae, Percidae, Esocidae and Lotidae. Brain AChE activity varied among fish species approximately 15-fold, ranging from 138 to 2011 micromol/g per h. All cyprinids had higher brain AChE activity than other fish families. Plasma AChE activity was on average 100-fold lower than that in brain, varying from 1.2 to 18.6 micromol/ml per h. Plasma BChE activity was found only in cyprinids with the exception of the common and crucian carp, and sabrefish. It varied from 26 to 1083 micromol/ml per h. In bream (Abramis brama) only 30% of specimens studied had BChE activity. The correlation coefficient values between activities of brain and plasma AChE, brain AChE and plasma BChE, plasma AChE and BChE were 0.67, 0.68 and 0.84, respectively. Cross-species and also cross-family differences in AChE and BChE activities among fish were demonstrated. Possible reasons for the differences are discussed.     

Acetylcholinesterase in Huntington''s and Alzheimer''s Diseases: Simultaneous Enzyme Assay and Immunoassay of Multiple Brain Regions

A newly developed enzyme-linked immunosorbent assay for acetylcholinesterase (AChE) protein was combined with conventional measures of enzyme activity in a study of 15 brain regions from six control cases (non-neurological deaths), six cases of Alzheimer's disease, and six cases of Huntington's disease. In the control brains, the mean AChE activity varied 100-fold from region to region (cortex lowest, striatum highest). The variation in enzyme activity was exactly paralleled by a variation in protein immunoreactivity. Overall, the homospecific activity of AChE averaged 0.26 +/- 0.007 mU/pg, close to the value for electrophoretically homogeneous enzyme isolated from red blood cells. Similar homospecific activities were observed in samples from Huntington's and Alzheimer's brains. Evidently, AChE that is immunoreactive but enzymatically inactive does not accumulate in any of the three conditions examined. Huntington's brain samples showed normal total contents of AChE, but Alzheimer's brains showed significant decreases of both enzyme activity and immunoreactivity in all seven cortical regions and in two out of the eight subcortical structures examined, hippocampus and nucleus accumbens.     

Quantitative succinate dehydrogenase histochemistry in the hippocampus of aged rats

Summary Quantitative histochemical methods (microphotometric kinetic and end-point measurements, and morphometric analyses of reactive areas) were used to investigate the levels of succinate dehydrogenase (SDH) in the hippocampus of young adult (3–6 months old) and aged male rats (24–27 months old). Methodological studies concerning the demonstration of SDH activity, which were performed using hippocampi of young animals, revealed a linear relationship between the reaction time and the amount of reaction product for up to 20 min; kinetic (continuous) and end-point measurements provided the same results. In a number of experiments, it was established that an incubation medium consisting of 100 mM succinate, 10 mM sodium azide, 3 mM nitro blue tetrazolium chloride, 0.25 mM phenazine methosulfate, and 7.5% polyvinylalcohol in 0.05M Hepes buffer (final pH 7.5) was optimal for quantitative SDH histochemistry in the hippocampus. Comparative quantitative investigations of SDH activity in rat hippocampi showed that, in most regions and layers of the hippocampus of both young and aged rats, the levels of SDH activity increased along the rostrocaudal axis of the hippocampus, i.e., higher levels were present in the caudal than in the rostral pole. In both groups, the highest SDH levels were observed in the molecular layer of the cornu ammonis (CA)-1 the CA-3, and the fascia dentata (middle and outer thirds), most of which are termination fields of the excitatory perforant path arising from the regio entorhinalis. Furthermore, in almost all of the investigated layers, the older animals exhibited lower SDH levels than young animals. These differences were statistically significant in the molecular layer of the fascia dentata and in most layers of the CA-3. The lower SDH levels in aged animals are discussed in relation to the reduced capacity for energy metabolism in the aging brain.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the Deutsche Forschungsmeinschaft (Ku 541/2-1)     

Effect of thermal stress and water deprivation on the acetylcholinesterase activity of the pig brain and hypophyses

The effects of direct exposure of boars to thermal stress for 1 h daily for 5 days and to acute water deprivation for 24 or 48 h were studied on the acetylcholinesterase (AChE) activity of porcine brain and hypophysial regions. Mean ambient temperatures, respiratory rates and rectal temperatures in the open were significantly higher than inside the pen. Heat stress induced a rise in AChE activities in the pons, cerebellum, amygdala, hippocampus, hypothalamus, mid-brain and medulla oblongata. However, no significant changes were observed in the cerebral cortex, adenohypophysis and neurohypophysis. Water deprivation significantly (P<0.05) depressed AChE activity to varying extents depending on the duration of water restriction. Thus AChE activity in the amygdala was depressed by water deprivation for 24 h but partially restored at 48 h. The pons and medulla oblongata were comparable to the amygdala in this respect. The adenohypophysis and neurohypophysis were relatively unaffected.     

Effect of acetylcholinesterase activity on pathogenesis of airway hyperresponsiveness in guinea pigs.

To clarify the effect of acetylcholinesterase (AChE) on the pathogenesis of airway hyperresponsiveness, AChE activities in tracheal smooth muscle and lung tissue from congenitally bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs were compared. For this purpose, AChE activities were determined by measuring the rate of absorbance of tissue homogenate. Relative amounts of AChE mRNA were also evaluated by the RT-PCR method. In both tracheal smooth muscle and lung tissue from BHS, the AChE activity and the relative amount of AChE mRNA were less than those in BHR. These results suggest that the reduced AChE activity is at least a candidate for inducing airway hyperresponsiveness.     

Changes in the acetylcholinesterase and choline acetyltransferase activities in the early development of the chick embryo

Summary Acetylcholinesterase (AChE, EC 3.1.1.7) and choline acetyltransferase (CAT, EC 2.3.1.6) activities where studied in the early development of the chick embryo. A sharp increase in AChE activity occurred in the gastrulating embryo. The highest AChE activity was associated with hypoblast cells. By sucrose density gradient centrifugation three molecular forms of AChE with sedimentation coefficients 4.7 S, 6.8 S and 10.9 S were determined. During the gastrulation there was no remarkable change in the activity of CAT. A two-fold decrease in the CAT activity occurred at the end of gastrulation.     

Distinct Changes in Peptide YY Binding to, and mRNA Levels of, Y1 and Y2 Receptors in the Rat Hippocampus Associated with Kindling Epileptogenesis

Abstract: Electrical kindling of the rat dorsal hippocampus induced significant changes in the binding of ¹²⁵I-peptide YY to Y1 and Y2 subtypes of neuropeptide Y receptors and in their mRNA levels in the area dentata as assessed by quantitative receptor autoradiography and in situ hybridization histochemistry. Binding to Y1 receptor sites decreased by 50% ( p < 0.05) in the molecular layer of the stimulated dentate gyrus, 2 days after preconvulsive stage 2 and 1 week or 1 month after generalized stage 5 seizures compared with sham-stimulated rats. Binding to Y2 receptor sites increased bilaterally by 36–87% ( p < 0.05) in the hilus at stage 2 and 1 week or 1 month after stage 5. No significant changes were observed after one afterdischarge or in the other hippocampal subfields or in the cortex. Y1 receptor mRNA signal decreased bilaterally by 50–64% ( p < 0.01) in the granule cell layer, 6 h but not 24 h after stages 2 and 5. The Y2 receptor mRNA signal was enhanced by 283% ( p < 0.01) in the stimulated granule cell layer 24 h after stage 2. At 6 and 24 h after stage 5, mRNA levels were increased both ipsilaterally (283 and 360%, respectively; p < 0.01) and contralaterally (190 and 260%, respectively; p < 0.05). No significant changes in level of either mRNA was found following one afterdischarge. These modifications, and the enhanced neuropeptide Y release previously shown in the hippocampus, suggest that kindling is associated with lasting changes in neuropeptide Y-mediated neurotransmission.     

Divergent Regulation of Acetylcholinesterase and Butyrylcholinesterase in Tissues of the Rat

Abstract: Investigating the possibility that acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are regulated in a coordinated manner, we have examined the natural variation in activity of these two enzymes in several tissues of adult male Sprague-Dawley, Fischer-344, and Wistar-Furth rats. Both enzymes varied greatly in mean activity among brain, diaphragm, atria, serum, superior cervical ganglia, and liver. In Sprague-Dawley rats there were also large individual variations with up to a fivefold range of AChE activities and up to a 100-fold range of BuChE activities in a given tissue. Individual variations in cholinesterase activities appeared to be smaller in the inbred Fischer-344 or Wistar-Furth rats. Experiments with internal standards of partially purified AChE and BuChE indicated that the individual variations probably reflected differences in the intrinsic content or specific activity of the tissue enzymes. Comparison of the AChE activities in different tissues of a given group of rats failed to reveal statistically significant correlations in any strain (i.e., the relative activity of any one tissue was no guide to the relative activity of any other tissue in the same rat). This result indicates that the regulation of AChE is tissue-specific. By contrast, BuChE activity showed highly significant correlations among the majority of the tissues examined in the Sprague-Dawley rats, implying that widely dispersed factors can affect the regulation of this enzyme. Body-wide regulation is not necessarily the rule, however, since only a single tissue pair in the inbred Fischer rats and none of the pairs in the Wistar-Furth rats showed significant correlations of BuChE activity. In general, AChE and BuChE activities were not correlated with each other to a statistically significant degree. We conclude that the control of these enzymes normally involves different mechanisms and is strongly affected by the genetic background of the sample population.     

THE CORRELATION BETWEEN CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE ACTIVITY IN DIFFERENT AREAS OF THE CEREBELLUM OF RAT AND GUINEA PIG

Abstract— Choline acetyltransferase (ChAc) and acetylcholinesterase (AChE) levels were measured quantitatively in samples from the archi- and paleocerebellar vermis (Larsell's Lobules IX c,d,-X, and Lobules VII-VIII, respectively) and from the cerebellar peduncles, nuclei and white matter of rat and guinea pig. Lesions to isolate archi- or paleocerebellar areas were made in some rats and the effect on enzyme levels and ultrastructure were studied. In the rat there was a striking correlation between the activity of ChAc and AChE in the different areas; thus in the archicerebellar cortex the levels of both enzymes were 3–4 times those in the paleocortex. Deafferentation caused a fall in ChAc and this practically paralleled the fall in AChE in the same area. The reduction in both enzymes was more pronounced in the archi- than in the paleocerebellar cortex. In the guinea pig the results were very different. The ChAc activity was much lower than in the rat and was equal in the archi- and paleocerebellum. The AChE activity was also uniform in the different areas but, in contrast to ChAc, was higher than in the rat.     

Enzymatic determination of choline acetyltransferase by coenzyme A cycling and its application to analysis of single mammalian neurons

Abstract: An enzymatic assay for choline acetyltrans-ferase was developed by measuring acetyl-coenzyme A (acetyl-CoA) formed from CoASH and acetylcholine (ACh). This method is extremely sensitive and may be applied to the analysis of microgram to nanogram crude samples. The method is, however, not useful when choline acetyltransferase is present in very low concentrations. The basis of this method is to amplify a small amount of synthesized acetyl-CoA in the assay mixture by using an enzymatic amplification reaction, CoA cycling. This amplification mechanism made it possible to perform microassays (13 nl-2.2 μl of assay volume) of freeze-dried sections prepared from cerebral cortex, striatum, and hippocampus of mice and single cell bodies isolated from freeze-dried sections of rabbit spinal cords. These samples were weighed and added directly to the reaction mixture. The activities of the above cerebral regions, assayed with 1,500–2,000-fold amplification, corresponded well to the results previously reported by other workers. The average activity of single anterior horn cells, determined with 64,000–420,000-fold amplification, was 40-fold higher than that of rabbit cerebral cortex, and the specific activities on a dry weight basis were widely distributed among individual neurons. No activity was detected in the noncholinergic dorsal root ganglion cells or in cerebellar cortex.     

Is Butyrylcholinesterase of the Rat CNS a Membrane-Bound Enzyme?

Abstract: The study of Arrhenius plots for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity from the rat brain and spinal cord revealed that in contrast to AChE, which exhibited biphasic Arrhenius plots with a distinct break (transition temperature) at about 16–18°C, BuChE showed no evidence of discontinuity and a higher activation energy in the physiological range of temperature. The results indicate lack of lipid-protein interaction in the case of BuChE of the CNS tissue. It is inferred that BuChE, in contrast to AChE, is not bound in any significant way to cellular membranes of the CNS tissue.     

Localization of choline acetyltransferase in laminae of the rat olfactory tubercle

Abstract: We report the distribution of choline acetyltransferase (ChAT) activity in the laminae of the rat olfactory tubercle. Within its posterior medial portion, the tubercle contains three parallel histological laminae that can be separated by cutting tangential sections from frozen tissue. ChAT was measured in homogenates of consecutive sections (16 μm) cut parallel to these laminae. The distribution of ChAT activity, as a function of tubercle depth, showed a broad peak centered at 500 μm from the ventral surface of the brain. Enzyme activity measured at this depth (85 pmol acetylcholine formed/μg protein/h) was 2 1/2 times greater than that measured in the outermost, plexiform, layer. Stereotaxic injections of kainic acid (1 μg in 1 μ1) made directly into the tubercle were used to eliminate intrinsic neurons. Three days after injection, histological examination revealed the almost total absence of neuronal cell bodies and the proliferation of glial cells. The greatest decreases in ChAT activity (50%) were seen at depths of 300–600 μm whereas no loss of activity occurred in the plexiform layer.