DISTRIBUTION OF ACETYLCHOLINE, CHOLINE, CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN REGIONS AND SINGLE IDENTIFIED AXONS OF THE LOBSTER NERVOUS SYSTEM

Abstract— Acetylcholine, its precursor (choline), and the enzymes of its biosynthesis and degradation (choline acetyltransferase and acetylcholinesterase, respectively) have been studied and quantified in extracts of several regions of the nervous system of the lobster and in single, isolated axons of identified efferent excitatory, efferent inhibitory and afferent sensory neurons. The choline acetyltransferase is a soluble enzyme similar to that from other species. The predominant acetylcholine-hydrolysing enzyme is largely membrane-bound and has been characterized as a specific acetylcholinesterase. A single peak of acetylcholinesterase activity can be detected upon velocity sedimentation analysis of Triton X-100-treated extracts of all regions of the nervous system. Choline acetyltransferase distribution parallels that of sensory neural elements, and its specific activity shows nearly a 500-fold difference from the richest to the poorest neural source. Acetylcholinesterase levels span only a 23-fold range, and activity is found in all neural regions, including those free of known sensory components. A radiochemical microassay for choline and acetylcholine in the range of 20–2000 pmol is described in detail. All 3 types of axons contain comparable levels of choline ( ca. 2 pmol/μg protein), but acetylcholine is asymmetrically distributed. Efferent axons contain no detectable acetylcholine, while sensory axons from abdominal muscle receptor organs have an average of 1·9 pmol/μg protein. Choline acetyltransferase is similarly distributed; sensory axons show at least 500-fold greater activity than efferent axons. Acetylcholinesterase is nearly uniformly distributed among the three types of fibres. These results are discussed in terms of a general view of transmitter accumulation in single neurons.     

CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN CULTURED BRAIN CELLS FROM CHICK EMBRYOS

—Dissociated cells from brains of 7-day chick embryos were grown in primary culture for as long as 20 days. Many of the plated cells grew out long processes. Others, which proliferated rapidly, formed a confluent layer of flat cells after 4-6 days. Total DNA and protein increased five-fold, and activity of choline acetyltransferase (EC2.3.1.6) increased about 40-fold in 20 days. Acetylcholinesterase (EC3.1.1.7) increased three-fold by the fourth day of culture and then declined. The pattern of increase for choline acetyltransferase was similar to that for the in vivo development of the enzyme. l -Thyroxine, cyclic AMP (adenosine-3′,5′-monophosphate) or theophylline promoted increased levels of both enzymes by 30-200 per cent. l -Thyroxine also increased the activity of acetylcholinesterase in vivo by 40 per cent. When overgrowth by flat cells was prevented by the addition of 10^-3m -5-flourouracil, there was a decrease in the activity of choline acetyltransferase and an increase in the activity of acetylcholinesterase in comparison to control activities. The addition of 10^-3m -morphine or cocaine produced a 30 per cent elevation in the activity of choline acetyltransferase, but this effect could be mimicked with equimolar concentrations of ammonium ion.     

OCCURRENCE AND DISTRIBUTION OF AROMATIC l-AMINO ACID (l-DOPA) DECARBOXYLASE IN THE HUMAN BRAIN

—The enzymatic decarboxylation of l -DOPA was measured in isotonic dextrose homogenates of different regions of the human brain by estimating ¹⁴CO₂ evolved from tracer amounts of d l -DOPA[carboxy1-¹⁴C]. Enzyme activity was linear with respect to tissue concentration and time of incubation. The reaction exhibited a pH maximum at 7·0, was completely dependent upon the presence of high concentrations of pyridoxal phosphate, proceeded at the same rate in an atmosphere of air and nitrogen, and produced dopamine in addition to CO₂ as a reaction product. The enzyme preparation behaved like an aromatic l -amino acid decarboxylase: it also decarboxylated o-tyrosine and when incubated with 5-hydroxytryptophan, serotonin was isolated as the reaction product; but it was devoid of activity towards d -DOPA[carboxy1-¹⁴C]. Within the human brain, l -DOPA decarboxylase was most active in the putamen and caudate nucleus; the pineal gland, hypothalamus, and the reticular formation and dorsal raphe areas of the mesencephalon exhibited considerable activity. Areas of cerebral cortex exhibited very low enzymatic activity and in regions composed predominantly of white matter, l -DOPA decarboxylase activity was not significantly above blank values. The activity of l -DOPA decarboxylase in the human putamen and caudate nucleus tended to decrease with the age of the patients; in comparatively young subjects (46 yr old) the enzyme activity compared favourably with that found, by means of the same assay technique, in the caudate nucleus of the cat.     

EFFECT OF PYRIDOXINE DEFICIENCY ON AROMATIC l-AMINO ACID DECARBOXYLASE IN THE DEVELOPING RAT LIVER AND BRAIN

—Maternal pyridoxine deficiency begun 2 weeks before mating and continued throughout pregnancy and the nursing period resulted in diminished wt. gains in the brain, the liver and the body in the first 16 days of life, as well as lowered levels of the aromatic l -amino acid decarboxylase in both brain and liver tissue. The fetus was protected from the effect of vitamin B₆ deficiency during pregnancy, since at birth the body wt., organ weights, and decarboxylase levels in these tissues were comparable to those of control litters. The brain was affected less than the liver, both in rate of wt. increase and decarboxylase activity. The cerebellum normally developed measurable decarboxylase activity only during the second week of life. The cortex normally slowly increased its low decarboxylase activity during the first week postnatally, with a more rapid increase during the second week. This rapid increase was primarily in the holoenzyme moiety. The rest of the brain, which had well developed levels of decarboxylase activity at birth, normally showed a sharp increase during the second week of life which was also largely in the holoenzyme portion. When the increasing weights of these tissues were considered, it became obvious that the total amount of apoenzyme as well as the amount of holoenzyme were increasing in the normally developing rat, although the greatest amount of the change was in the holoenzyme form. The liver normally showed a much more rapid increase in decarboxylase activity than did the brain, and showed the increase much earlier. The holoenzyme normally increased rapidly after the first 4 days, whereas the apoenzyme concentration levelled off at this time. The effect of the pyridoxine deficiency on decarboxylase activity was almost entirely on the holoenzyme form of the decarboxylase, since the apoenzyme form generally remained the same in the control and the deficient pups during development. There appeared to be no decarboxylase inhibitor present in pyridoxine deficient tissues, nor any evidence in control tissues for an enzyme required for the activation of the decarboxylase by cofactor.     

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

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

TOPOGRAPHICAL AND SUBCELLULAR DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND GLUTAMATE DECARBOXYLASE IN PIGEON OPTIC TECTUM

Abstract— The distribution of choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) in different layers of the pigeon optic tectum and in some nuclei of the optic lobe have been investigated. About 40% of GAD and 25% of ChAT were found in the superficial part of tectum, but negligible activity was found in the stratum opticum. The highest GAD activity was found in layers 3-7 (according to the nomenclature of C ajal , 1911) with a peak in layer 4. ChAT activity peaked in layers 3, 5. 8 and 10/11. Its distribution correlated well with the staining pattern of AChE, particularly in the superficial part of the tectum. The distribution of ChAT and GAD did not change significantly 4 weeks after enucleation. ChAT and GAD activities were high in the nucleus isthmi, pars parvocellularis (Ipc). The activity of GAD was also high in the nucleus intercollicularis (ICo), the other nuclei showed less activity of both enzymes.     

THE CHARACTERIZATION OF HISTIDINE DECARBOXYLASE AND ITS DISTRIBUTION IN NERVES, GANGLIA AND IN SINGLE NEURONAL CELL BODIES FROM THE CNS OF APLYSIA CALIFORNICA

Histamine (HA) is present in substantial quantities in all ganglia of Aplysia californica. Within the cerebral ganglia this amine is known to be concentrated in at least two identified neurons designated C-2 neurons. In this study a combination of chemical and enzymatic analyses was employed to provide evidence for the existence of a biochemical pathway for HA synthesis in ganglia and individual neurons of this marine mollusk. Examination of extracts of individual neurons dissected from ganglia organ-cultured in the presence of [³H]histidine showed that every neuron accumulated labelled histidine, but only the HA-containing C-2 neurons synthesized and stored labelled HA suggesting that the formation of HA in Aplysia could be catalyzed by the enzyme histidine decarboxylase (HDC). HDC activity was studied with a new microradiometric assay. Many of the properties of the molluscan HDC studied were found to correspond to the vertebrate enzyme. Enzyme activity was inhibited by α-hydrazino-histidine but unaffected by concentrations of α-methyldopa or by 5-(3,4-dihydroxycinnamoyl) salicylic acid which produced nearly complete inhibition of aromatic amino acid decarboxylase activity. HDC was measurable in nervous but not other Aplysia tissues assayed. All 5 major ganglia contained HDC activity which spanned a 15-fold range between the least and most active ganglia. Only 4 of the 13 nerve trunks assayed yielded measurable enzymic activity; these active nerves were associated with the cerebral ganglia which has the highest HDC activity of all measured ganglia. Of the numerous individual neurons assayed for HDC, only the C-2 cells showed measurable enzyme activity, about 25 pmol/cell/h or 70 μmol/g protein/h. Since the activity of HDC in the HA-containing neurons was at least three orders of magnitude larger than all other neurons assayed in the cerebral and other ganglia, these data appear to provide a direct metabolic basis for the selective presence of HA in these cells, and they indicate that the cellular presence of HDC provides a useful biochemical marker for the location of HA-rich neurons in Aplysia.     

ACETYL-CoA SYNTHESIZING ENZYME ACTIVITIES IN SINGLE NERVE CELL BODIES OF RABBIT

Activities of five enzymes (pyruvate dehydrogenase complex; citrate synthase, EC 4.1.3.7; carnitine acetyltransferase, EC 2.3.1.7; acetyl-CoA synthetase, EC 6.2.1.1; and ATP citrate lyase, EC 4.1.3.8) were determined in cell bodies of anterior horn cells and dorsal root ganglion cells from the rabbit. For comparison, molecular layer, granular layer and white matter from rabbit and mouse cerebella and cerebral cortex and striatum from the mouse were analyzed. Samples (3–85 ng dry weight) were assayed in 180 to 370 ml of assay reagents containing CoASH and other substrates in excess. By using ‘CoA cycling’, the assay systems were devised to amplify and measure small amounts of acetyl-CoA formed during the enzyme reactions. Carnitine acetyltransferase was the most active enzyme in single nerve cell bodies and all layer samples, except for rabbit and mouse cerebellar white matter. Citrate synthetase was the lowest in single cell bodies. The activities of carnitine acetyltransferase and acetyl-CoA synthetase (656 and 89.8 mmoles of acetyl-CoA formed/kg of dry weight/h at 38°C) from dorsal root ganglion cells were about 2-fold higher than those from anterior horn cells. The activity of ATP citrate lyase (134mmol of acetyl-CoA formed/kg of dry weight/h at 38°C) from anterior horn cells was approximately twice that from dorsal root ganglion cells. The activity of this enzyme was distributed in a wider range in anterior horn cells than dorsal root ganglion cells. The second highest activity (80.0 mmol of acetyl-CoA formed/kg of dry weight/h at 38°C) of ATP citrate lyase was found in striatum where cholinergic interneurones are abundant. Relatively higher activities of this enzyme were found in cerebellar granular layer and white matter which are known to contain the cholinergic mossy fibers. These results suggested that cholinergic neurones contain higher activity of ATP citrate lyase which is thought to supply acetyl-CoA to choline acetyltransferase (EC 2.3.1.6) as a substrate to form acetylcholine.     

AXOPLASMIC TRANSPORT OF AROMATIC l-AMINO ACID DECARBOXYLASE (EC 4.1.1.26) AND DOPAMTNE β-HYDROXYLASE (EC 1.14.2.I) IN RAT SCIATIC NERVE

The axoplasmic transport of aromatic l -amino acid decarboxylase and dopamine β-hydroxylase, two enzymes involved in the biosynthesis of catecholamines, was studied in rat sciatic nerve. The two enzymes exhibited markedly different axoplasmic flow characteristics, since dopamine β-hydroxylase activity accumulated on the proximal side of a ligation nearly three times as fast as aromatic l -amino acid decarboxylase activity. Distally dopamine β-hydroxylase activity remained essentially constant for 24 h, whereas aromatic l -amino acid decarboxylase activity fell precipitously. Evidence was obtained to rule out the possibility that differences in the rate of inactivation of the two enzymes could account for the different rates of accumulations observed. The conclusion, that aromatic L-amino acid decarboxylase and dopamine β-hydroxylase are transported in sympathetic nerve at different rates is discussed in relation to the biosynthesis of norepinephrine.     

DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND GLUTAMATE DECARBOXYLASE WITHIN THE SUBSTANTIA NIGRA AND IN OTHER BRAIN REGIONS FROM CONTROL AND PARKINSONIAN PATIENTS

—The distribution of choline acetyltransferase (ChAc, EC 2.3.1.6) and l -glutamate 1-carboxylyase (glutamate decarboxylase, GAD, EC 4.1.1.15) was studied in serial frontal slices of the substantia nigra (SN) (pars compacta, PC; pars reticulata, PR; an intermediate region, IR) as well as in other brain areas from post mortem tissue of control and Parkinsonian patients. Within the SN from control brain ChAc and GAD activities showed a distinctive distribution: ChAc activity in PC was higher than in PR and IR by 427% and 253% respectively and within PC the enzyme activity in the rostral part exceeded that in the control part by 353%. The GAD activity in PC was higher by 41% than that in PR and within PC seemed to be higher in the caudal than in the rostral part. For both enzyme activities there were no significant differences between PR and IR or within these regions. In Parkinsonian brain both ChAc and GAD activities were reduced to 15-25% of controls in all 3 regions of the SN. The distinctive distribution of ChAc and GAD activity found in the SN of control brain was abolished: no difference was observed between the 3 regions. However, within PC the ChAc activity was lower in the medial than in the rostral part. Since nigral ChAc is possibly located in interneurons, the decrease in enzyme activity may be connected with the cell loss observed in the SN of Parkinsonian brain. By contrast, nigral GAD is probably contained in terminals of strio-nigral neurons and the decrease in enzyme activity in Parkinson's disease in the absence of striatal cell loss, may reflect a change in the functional state of these GABA neurons. Among various areas of control brains ChAc activity was highest in caudate nucleus and putamen while GAD was highest in SN. caudate nucleus, putamen and cerebral cortex. In Parkinsonian brain the most severe reduction in ChAc and GAD activities was found in the SN.     

REGIONAL CHANGES IN RAT BRAIN CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE ACTIVITY RESULTING FROM UNDERNUTRITION IMPOSED DURING DIFFERENT PERIODS OF DEVELOPMENT1

Abstract— The severity of mental changes in malnourished children is related to both the period of development when the malnutrition occurs and the amount of environmental stimulation. In the present study the effect of imposing protein undernutrition during the period of gestation or postweaning period, and protein-energy undernutrition during the suckling period on cholinergic enzyme activity was investigated in the rat. Six different dietary treatments were given and the activity of ChAc, ChE, and AChE determined in the forebrain, brainstem, and cerebellum of male rats on day 49. Undernutrition imposed during gestation, suckling or postweaning all resulted in changes in cholinergic enzyme activity. The direction and degree of change of enzyme activity depended on the period when undernutrition was imposed as well as the brain region. In the forebrain ChE and AChE activities were altered, in the brainstem, ChAc, ChE and AChE activities were altered, and in the cerebellum ChAc activity was altered. The effect on the activity of the individual cholinergic enzymes was complex and was not the same in the different regions of the brain or even for the same brain region exposed to undernutrition during different periods of development. These results along with earlier work indicate that cholinergic enzyme activity in brain of undernourished rats can be altered by both the period of development when undernutrition is imposed and the amount of environmental stimulation.     

SNAIL FARMING: A SURVEY OF BREEDING MANAGEMENT, HYGIENE AND PARASITISM OF THE GARDEN SNAIL, HELIX ASPERSA MULLER

Fifteen farms in 1984 and twenty in 1985 were investigated forbreeding management, hygiene and parasitism. Farm hygiene didnot seem to play an important role on the breeding performancesin our samples. Nematodes were the most frequent parasites:Alloionema appendiculatum were equally prevalent among juvenileand adult snails whereas Angiostoma aspersa and Nemhelix bakeriwere found mostly in reproductive adults. The presence of nematodes,assessed by coproculture, was negatively related to breedingperformances     

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.     

NATURAL SELECTION REDUCES ENERGY METABOLISM IN THE GARDEN SNAIL, HELIX ASPERSA (CORNU ASPERSUM)

Phenotypic selection is widely recognized as the primary cause of adaptive evolution in natural populations, a fact that has been documented frequently over the last few decades, mainly in morphological and life-history traits. The energetic definition of fitness predicts that natural selection will maximize the residual energy available for growth and reproduction, suggesting that energy metabolism could be a target of selection. To address this problem, we chose the garden snail, Helix aspersa ( Cornu aspersum ). We performed a seminatural experiment for measuring phenotypic selection on standard metabolic rate (SMR), the minimum cost of maintenance in ectotherm organisms. To discount selection on correlated traits, we included two additional whole-organism performance traits (mean speed and maximum force of dislodgement). We found a combination of linear (negative directional selection, β=−0.106 ± 0.06; P = 0.001) and quadratic (stabilizing selection, γ=−0.012 ± 0.033; P = 0.061) selection on SMR. Correlational selection was not significant for any possible pair of traits. This suggests that individuals with average-to-reduced SMRs were promoted by selection. To the best of our knowledge, this is the first study showing significant directional selection on the obligatory cost of maintenance in an animal, providing support for the energetic definition of fitness.     

VARIABILITY OF EGG CANNIBALISM IN THE LAND SNAIL HELIX ASPERSA IN RELATION TO THE NUMBER OF EGGS AVAILABLE AND THE PRESENCE OF SOIL

Egg cannibalism by hatchlings has been demonstrated in somepulmonate land snails; this behaviour is promoted by a highhatching asynchrony within the egg-batch. Under laboratory conditions,the percentage of new-born snails Helix aspersa having cannibalisedunhatched eggs was not influenced by the soil factor: about70% of them ingested one egg within their first four days oflife whether soil was present or not. The propensity to eggcannibalism in hatchlings of H. aspersa increased with egg density.However, most of the new-born hatchlings consumed a single eggduring the four days following hatching, and only exceptionallytwo. The consumption of one egg increased the snails wet weightby 38.7% within four days. A weak ingestion of soil componentsalso occurred, but it induced a growth that was three-timesless than that due to the consumption of an egg. In addition,the survival of newly hatched snails maintained under non-dehydratingthermohygrometric conditions was high, even when they were submittedto four days food-deprivation. (Received 22 July 1999; accepted 24 November 1999)     

ACETYLCHOLINE,CHOLINE AND CHOLINE ACETYLTRANSFERASE ACTIVITY IN THE DEVELOPING BRAIN OF NORMAL AND HYPOTHYROID RATS1

Abstract— Acetylcholine, choline and choline acetyltransferase activity were measured in the whole brains of normal and hypothyroid rats during development. At 1 day postpartum, brain acetylcholine was 73 per cent of adult levels. Propylthiouracil-induced hypothyroidism up to age 20 days did not alter brain acetylcholine concentrations, but at 30 days resulted in significantly decreased levels. At day 1, brain choline was 20 per cent higher than adult levels and decreased between days 8 and 10. In hypothyroid rats this phenomenon did not occur until days 15–20. At day 1 postnatally, choline acetyltransferase activity was only 7 per cent of adult levels, then between days 5 and 20 rose to 77 per cent of adult levels. Beginning at day 8, hypothyroidism resulted in significantly decreased enzyme levels. This effect could be reversed at day 17 by concurrent tri-iodothyronine substitution therapy. In hypothyroid rats, maximum brain choline acetyltransferase activity was 30 per cent less than normal adult levels.     

THE SYNTHESIS OF ACETYLCHOLINE FROM ACETYL-CoA, ACETYL-DEPHOSPHO-CoA AND ACETYLPANTETHEINE PHOSPHATE BY CHOLINE ACETYLTRANSFERASE

Abstract— The synthesis of ACh by choline acetyltransferase (ChAc) has been examined using acetyl-CoA, acetyl-dephospho-CoA and acetylpantetheine phosphate. At pH 7.5 K_m values of 25.7 μ m for acetyl-CoA, 54.8 μ m for acetyl-dephospho-CoA and 382 μ m for acetylpantetheine phosphate were obtained and are similar to those at pH 6.0. This indicates that the 3-phosphate may not be required for binding the substrate to the enzyme unlike carnitine acetyltransferase.
Inhibitor constants ( K_i ) for CoA, dephospho-CoA and pantetheine phosphate were also measured and when considered with the K_m values obtained for the acetyl derivatives it is concluded that acetyl-dephospho-CoA could be a successful acetyl donor in the synthesis of ACh.
Acetyl-dephospho-CoA was found to be less satisfactory as a substrate for citrate synthase.