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.     

Subregional and Intracellular Distribution of NADP-Linked Malic Enzyme in Human Brain

High total activity (expressed as μmol/min/g of wet tissue or per milligram of DNA) and differential subregional distribution of NADP-linked malic enzyme was found in autopsy specimens of human brain. Striatum showed the highest activity of malic enzyme, which was two to five-fold higher than that in other human organs tested. High activity was also found in frontal cortex, while the lowest activity of the enzyme in the central nervous system was found in cerebellum, substantia alba, and corpus callosum. In striatum, frontal cortex, pens, and cerebellum more than 80% of total malic enzyme activity was localized in the mitochondrial fraction, while in substantia alba and corpus callosum approximately 60% of the enzyme activity was present in the mitochondrial fraction. Relatively high specific activity of malic enzyme was found in a crude mitochondrial fraction isolated from various regions of human brain. The highest specific activity was found in the mitochondria isolated from striatum (more than 100 nmol/min/mg of mitochondrial protein); the lowest, but still high (approximately 32 nmol/min/mg of mitochondrial protein) was present in corpus callosum. These data and the different ratios of citrate synthase to mitochondrial malic enzyme activities found in different regions of brain suggest that human brain mitochondria, like the mitochondria isolated from other mammalian brains, are extremely heterogenous. A possible role of mitochondrial malic enzyme in human brain metabolism is discussed.     

Structural neuroimaging in autism

Structural neuroimaging studies done by means of magnetic resonance imaging (MRI) have provided important insights into the neurobiological basis for autism. The aim of this article is to review the current state of knowledge regarding structural brain abnormalities in autism. Results of MRI studies dealing with total brain volume, the volume of the cerebellum, caudate nucleus, thalamus, amygdala, hippocampus and the area of the corpus callosum are summarized. Existing research suggests that autistic individuals have larger total brain, cerebellar and caudate nucleus volumes; however, the area of the corpus callosum is reduced. Results of studies involving the amygdala and hippocampus volume in autistic subjects remain inconsistent and no changes have been detected in thalamic volume.     

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 and subcellular distribution of choline acetyltransferase in the brain of rats

—Homogenates of corpus striatum, cerebral cortex and hypothalamus excised from rat brain were fractionated on discontinuous Ficoll and sucrose density gradients, and the distribution of choline acetyltransferase (ChAc) in the mitochondrial and synaptosomal fractions was determined. In the hypothalamic and cortical regions the fractions enriched in synaptosomes showed much higher activity of ChAc than those containing mainly mitochondria. On the other hand, the corpus striatum showed an equal distribution of ChAc activity in those two fractions. The localization of ChAc was also studied in the postnuclear supernatants obtained from three brain regions, using continuous sucrose density gradients. The distribution of ChAc was compared to that of monoamine oxidase (MAO), potassium and protein. When the pellets obtained from the fractions collected from the gradient were suspended in sucrose, the peak of ChAc activity was close to that of MAO in all three brain regions. When 0.1 mm EDTA +1% butanol was used in order to liberate the occluded form of ChAc, the maximum liberation occurred in lighter fractions, resulting in a shift of the activity peak toward the top of the gradient. This was found with fractions from hypothalamic and cortical regions. In the striatum, the liberated ChAc remained in the same fractions as the occluded enzyme. The results indicate that ChAc is liberated only in those fractions where it is present in synaptosomes. In agreement with the results on the discontinuous gradients this occurs in particles of lower density than mitochondria in cortex and hypo-thalamus, but in particles of similar density to mitochondria in the corpus striatum, indicating regional differences in the distribution of ChAc in the brain. K+ containing particles centrifuged in less dense fractions than those containing ChAc, indicating that synaptosomes are heterogeneous with respect to these two marker substances.     

THE EFFECTS OF PENTOBARBITONE-SODIUM ANAESTHESIA, SHOCK AVOIDANCE CONDITIONING AND ELECTRICAL SHOCK ON ACETYLCHOLINESTERASE ACTIVITY AND PROTEIN CONTENT IN REGIONS OF THE RAT BRAIN

Abstract— Pentobarbitone sodium anaesthesia was found to produce an increase in protein content in some regions of the rat brain, i.e. posterior cortex, caudate nucleus, and a decrease in protein content in the ventral cortex.
Acetylcholinesterase expressed in terms of wet weight was found to increase in the cerebellum, medulla, and to decrease in the medial cortex, hippocampus, thalamus and caudate nucleus. The changes in activity were not explicable in terms of a direct effect of the anaesthetic on the enzyme. A decrease in protein content of rat brain was observed in the frontal cortex, ventral cortex, hippocampus and caudate nucleus after electrical shocks. Following shock avoidance conditioning procedure (shuttle-box), decreases in protein content were observed in the medial cortex, posterior cortex, cerebellum and ventral cortex; in the thalamus an increase in protein content was observed.
Changes in AChE activity were observed following footshock in the frontal cortex and medulla where there was an increase in activity and in the caudate nucleus, hypothalamus, thalamus, and olfactory tubercle where there was a decrease in activity.
Following shock avoidance conditioning the activity of the AChE increased in posterior cortex, hippocampus, thalamus and hypothalamus and the activity of the enzyme decreased in the ventral cortex.     

Modulation in Acetylcholinesterase of Rat Brain by Pyrethroids In Vivo and an In Vitro Kinetic Study

Abstract: The modulation in acetylcholinesterase (AChE) of rat brain by two pyrethroids—permethrin (PM) and cypermethrin (CPM)—was studied both in vivo and in vitro. PM inhibited AChE activity in all regions of the rat brain (cerebral cortex, cerebellum, corpora striata, brainstem, hippocampus, and hypothalamus) at 4, 8, and 12 h after gastric intubation, whereas CPM elevated the enzyme activity in vivo. Substrate-dependent enzyme kinetic studies have shown that PM and CPM behave as mixed-type inhibitors, as evidenced by alterations in both Michaelis-Menten constant ( K _m) and maximal velocity ( V _max) values. This indicates that both PM and CPM and substrate acetylcholine interact at hydrophobic subsites and may be able to bind simultaneously to the enzyme.     

The distribution of cytochrome-oxidase activity in rabbit brain

1. The cytochrome-oxidase activity in eleven structures of the rabbit brain has been investigated. 2. Kinetic data suggest that the same enzyme occurs in all brain structures but that the activity on a tissue-weight basis differs from structure to structure, being in general higher in the phylogenetically newer structures. 3. When the kinetic data are related to cell density (as measured by DNA concentration) all the neuron-containing structures of the cerebrum and brain stem show the same activity but the corpus callosum and the cerebellum show much less. 4. A special study was made of the distribution of activity and cell density within the diencephalon. 5. The results suggest that all neurons in the cerebrum and brain stem have a similar cytochrome-oxidase activity, which is about 80 times that of glia, and that the cerebellum is relatively richer in glia or that it contains neurons with a much lower cytochrome-oxidase activity, or both.     

Circadian variation in the acetylcholinesterase activity of specific rat brain areas

Abstract

Acetylcholinesterase (AChE) activity of the adenohypophysis, cerebellum, cerebral cortex, hypothalamus, amygdala, hippocampus, midbrain, pons, medulla oblongata and caudate nucleus was determined by a spectro‐photometric method in adult, male rats adapted toan LD 12:12cycle. Results of the study show that AChE activity is highest during the light phase and lowest during the dark phase of the cycle in all the brain areas studied except the adenohypophysis, cerebellum, hippocampus and hypothalamus. These findings expand earlier observations on the circadian variation in rat brain AChE activity and suggests a relationship with reported circadian variation in the acetylcholine levels of rat brain.     

THE REGIONAL AND SUBCELLULAR DISTRIBUTION OF CATECHOL-O-METHYL TRANSFERASE IN THE RAT BRAIN

Catechol-O-methyl transferase (COMT) activities determined in different regions of rat brain showed small variations. Highest activities were found in the hypothalamus and corpora quadrigemina, and lowest activities in the hippocampus and corpus striatum. The regional distribution of COMT was thus at variance with the distribution of DOPA decar- boxylase in this study and with the distribution of catecholamines and tyrosine hydroxylase reported in the literature. Determinations of the subcellular distribution of COMT in rat forebrain showed that 50 per cent of the activity was recovered in the high speed supernatant fluid and about 33 per cent in the crude mitochondrial fraction. Further separation of the latter by discontinuous sucrose gradients showed that the particulate COMT was found in the synaptosomal fraction in an occluded form. Full enzyme activity was only obtained after treatment with a detergent or after resuspension in water. After hypo-osmotic rupture of the crude mitochondrial fraction, COMT was recovered in the cytoplasmic fraction. The subcellular distribution of COMT was very similar to the ones of lactate dehydrogenase and DOPA decarboxylase. The proportions of soluble COMT obtained from homogenates of various regions of the brain differed from that of choline acetyl transferase and DOPA decarboxylase but were similar to that of lactate dehydrogenase. In conclusion, COMT is a cytoplasmic enzyme almost evenly distributed in the CNS. Its distribution does not resemble the distributions of the catecholamines or of the enzymes participating in the synthesis of catecholamines.     

Estimation of cholinesterase and choline acetyltransferase in bovine anterior pituitary, posterior pituitary, and pineal body

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were estimated colorimetrically with thiocholine (SCh) esters as substrates in homogenates of bovine anterior pituitary (AP), posterior pituitary (PP), and pineal body (PB), and the levels were referred to those of whole rat brain. The levels of BuChE were very similar in all four tissues, approximately 10.25 μm -BuSCh hydrolysed/g tissue/hr; indicating that this enzymic activity represents a common structural component, perhaps vascular elements. Acetyl-thiocholine (ASCh) hydrolysis by AChE for brain, PP, PB, and AP was 338, 37,24, and 6 μm /g/hr, respectively. Choline acetyltransferase (ChAc) was estimated by the formation of [¹⁴C]acetylcholine from [¹⁴C]acetyl CoA. ChAc activity of posterior pituitary was generally found to be 15–20 per cent that of brain; the activity was always lowest in the anterior pituitary and pineal body, sometimes undetectable, but generally 5–10 per cent that of brain. The basis for the interpretation that cholinergic components in the posterior pituitary are due to acetylcholme-containing nerve endings and in anterior pituitary and pineal body to axons of sympathetic neurons was discussed.     

Huntington's disease: regional alteration in muscarinic cholinergic receptor binding in human brain.

Huntington's Disease, an autosomal dominant neurological disorder, is characterized by diffuse neuronal degeneration particularly in the basal ganglia and cerebral cortex. The purpose of this study was to examine various discrete regions of choreic and control brains for alterations in muscarinic cholinergic receptor binding and choline acetyltransferase (ChAc) activity. Nine postmortem brains, three from patients with Huntington's Disease and six controls, were dissected into 17 discrete regions. Each regional homogenate was assayed for muscarinic receptor concentration by measuring specific membrane binding of [³H]-QNB, a potent muscarinic antagonist which selectively labels brain muscarinic receptors. Aliquots from each brain region were also assayed for ChAc activity. Of significance was the marked reduction in specific [³H]-QNB receptor binding in the caudate nucleus, putamen and globus pallidus of choreic brain while no significant alterations were detected in other brain regions. Significant decreases in ChAc activity were found in the caudate nucleus, putamen, and globus pallidus with no alterations in ChAc activity in the rest of the brain regions examined. The tissues were chosen such that protein levels were similar in both choreic and normal brain samples. The apparent reduction in the number of muscarinic cholinergic receptors in the choreic brains suggests that treatment with cholinomimetic drugs might be beneficial in Huntington's Disease.     

TRANS-SULPHURATION IN PRIMATE BRAIN: REGIONAL DISTRIBUTION OF METHIONINE-ACTIVATING ENZYME IN THE BRAIN OF THE RHESUS MONKEY AT VARIOUS STAGES OF DEVELOPMENT

—The regional distribution of methionine-activating enzyme (ATP:l-methionine S-adenosyltransferase; EC 2.4.2.13) in the brain of the Rhesus monkey was determined at various stages of development. Activity of the methionine-activating enzyme was highest in pituitary gland, cerebellum and occipital grey matter, and lowest in areas rich in white matter: spinal cord, subcortical white matter, corpus callosum and optic chiasm. There was no marked change in activity in any area during development from the first-trimester foetus to the juvenile animal. During the same period of development, activity of the methionine-activating enzyme in the liver increased approximately four-fold. The findings are discussed in relation to those transmethylating enzymes and/or methylated products which have been studied in mammalian brain. The presence of high activity of the methionine-activating enzyme in cerebellum and occipital grey matter suggests that previously unrecognized methylating processes may be important in the metabolism of these areas of brain.     

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.     

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.     

Immunochemical studies of mammalian brain acetylcholinesterase.

Abstract— Specific antibodies were raised in rabbits to acetylcholinesterase (AChE) from bovine caudate nucleus and the‘native’(14S + 18S) and globular (11S) forms of AChE from eel electric tissue. All AChE preparations were purified by affinity chromatography to a specific activity of 100–400 mmol acetylthiocholine hydrolyzed/mg protein/h. Antigenic specificities of the different enzyme forms were studied by immunodiffusion, Immunoelectrophoresis and micro-complement fixation. Minor differences in antigenic determinants were observed between the different molecular forms of electric tissue AChE. In crossover experiments using both eel AChE and bovine caudate AChE antisera there was complete absence of cross reactivity between the mammalian brain AChE and the different molecular forms of the electric tissue enzyme. Brain AChE activity was inhibited up to 50% in the presence of its antiserum.     

TRANSPORT, TURNOVER AND DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLIN-ESTERASE IN THE VAGUS AND HYPOGLOSSAL NERVES OF THE RABBIT

Abstract— The transport, distribution and turnover of choline O -acetyltransferase (ChAc, EC 2.3.1.6) and acetylcholinesterase (AChE, EC 3.1.1.7) in the vagus and hypoglossal nerves were studied in adult rabbits. The enzymes accumulated proximally and distally to single and double ligatures on both nerves and thus indicated both a proximo-distal and retrograde flow of the enzymes. Double ligature experiments indicated that only 5–20 per cent of the enzymes were mobile in the axon. The rate of accumulation of both enzymes above a single ligature corresponded to the slow rate of axonal flow provided that all the enzymes were mobile, but to an intermediate or fast flow if only a small part of the enzymes was transported. The distribution of ChAc along the hypoglossal neurons was studied and only 2 per cent of ChAc was confined to cell bodies, 42 per cent was localized to the main hypoglossal nerve trunks and 56 per cent to the preterminal axons and axon terminals in the tongue. The ratio of AChE to ChAc was about 3 in the hypoglossal nerve and 32 in the vagus nerve.
Transection of the hypoglossal nerve was followed by a decrease in the activity of ChAc in the hypoglossal nucleus and nerve and in the axons and their terminals in the tongue. The activity of AChE decreased in the hypoglossal nucleus and nerve but not in the tongue. The half-life of ChAc in preterminal axons and terminals of the hypoglossal nerve was estimated to be 16-21 days from the results obtained on transport, axotomy and distribution of the enzyme. Intracisternal injection of colchicine inhibited the cellulifugal transport of both enzymes and led to an increase in enzyme activity in the hypoglossal nucleus.     

Biochemical Characterisation and Localization in Brain of a Human Brain-Leucocyte Membrane Glycoprotein Recognised by a Monoclonal Antibody

Abstract: The F10-44-2 monoclonal antibody was originally shown to interact with a determinant found predominantly in human brain and leucocytes. In this study we demonstrate by quantitative absorption analysis with homogenates of the head of the caudate nucleus, putamen, thalamus, cerebral grey matter, cerebral white matter, corpus callosum and cerebellar folia that the determinant is restricted to the white matter of the CNS. Immunofluorescence studies on frozen sections of the above brain subregions confirm the absorption analyses, showing staining only of white matter. In addition, and unexpectedly, we found very bright staining around blood vessels, particularly in the cerebellum. Biochemical studies established that the molecule in white matter bearing the F10-44-2 determinants is a sialylated membrane glycoprotein with an apparent molecular weight of 90,000, which is similar to but slightly smaller than the T lymphocyte form of the antigen. Developmental studies comparing 16-week foetal and adult cerebrum showed a fivefold increase in F10-44-2 antigen content. Thus, in the human CNS, the F10-44-2 antigen is a medium-sized glycoprotein which is restricted to white matter and shows a marked increase in concentration during development. No such molecule has been described previously.     

Selective Inhibition of Acetylcholinesterase in the Cerebellum and Hippocampus of Mice Following an Acute Treatment with Malathion

Adult male ICR mice were treated by intraperitoneal injection with 250?mg/kg of bodyweight of commercial malathion (a dose corresponding to 1/12 the LD50). After 6?h, acetylcholinesterase (AChE) activity in blood, liver, and six brain regions was determined. A statistically significant inhibition was observed in whole blood (23%), liver (21%), and, in particular, the central nervous system; the greatest degree of AChE inhibition was observed in the cerebellum (45%), followed by the hippocampus (29%). There was no significant change in AChE activity in the caudate putamen, frontal cortex, midbrain, or pons medulla. These results demonstrate that the magnitude of AChE inhibition in peripheral tissues does not accurately reflect the central-inhibitory effects of malathion on AChE activity in specific brain regions.     

Changes in regional cerebral metabolism during systemic hyperthermia in humans.

Whole body hyperthermia may produce vasodialation, nausea, and altered cognitive function. Animal research has identified brain regions that have important roles in thermoregulation. However, differences in both the cognitive and sweating abilities of humans and animals implicate the need for human research. Positron emission tomography (PET) was used to identify brain regions with altered activity during systemic hyperthermia. Human subjects were studied under cool (control) conditions and during steady-state hyperthermia induced by means of a liquid-conditioned suit perfused with hot water. PET images were obtained by injecting [(18)F]fluorodeoxyglucose, waiting 20 min for brain uptake, and then scanning for 10 min. Heating was associated with a 23% increase in resting metabolic rate. Significant increases in cerebral metabolic rate occurred in the hypothalamus, thalamus, corpus callosum, cingulate gyrus, and cerebellum. In contrast, significant decreases occurred in the caudate, putamen, insula, and posterior cingulum. These results are important for understanding the mechanisms responsible for altered cognitive and systemic responses during hyperthermia. Novel regions (e.g., lateral cerebellum) with possible thermoregulatory roles were identified.