Effects of the histamine H3 agonist (R)-alpha-methylhistamine and the antagonist thioperamide in vitro on monoamine oxidase activity in the rat brain.

The effects of an H3 agonist, (R)-alpha-methylhistamine (alpha-MeHA), and an H3 antagonist, thioperamide, on monoamine oxidase (MAO) activity in rat hypothalamus were studied in vitro. Thioperamide was more potent in inhibiting MAO-B than MAO-A activity; MAO-B activity in rat hypothalamic homogenates was competitively inhibited by thioperamide with a Ki value of 175 micronM. From this in vitro experiment, the conversion of N-telemethylhistamine to N-tele-methylimidazoleacetic acid may be inhibited by thioperamide, suggesting that thioperamide may affect the regulation of histamine metabolism within histaminergic neurons. In contrast with the results obtained with thioperamide, alpha-MeHA inhibited MAO-A more potently than MAO-B activity; the Ki values for MAO-A and -B of hypothalamic homogenates were estimated to be 1.1 and 3.3 mM, respectively. The weak inhibitory effect of alpha-MeHA for MAO-B does not seem to be a major cause of changes in N-tele-methylhistamine concentrations.

Insulin-like growth factor-1 receptors in human spinal cord: changes in amyotrophic lateral sclerosis.

Neurotrophic factors are important for neuronal survival and maintenance in the adult nervous system. The regional distribution of insulin-like growth factor-1 (IGF-1) receptors in human spinal cords from controls and amyotrophic lateral sclerosis (ALS) patients was studied by immunohistochemistry and quantitative autoradiography. When comparing 125I-IGF-1 binding in the different spinal levels of normal spinal cord the same distribution pattern was found in which the binding was highest in the central canal > dorsal horn > ventral horn > white matter. In the ALS cases although a general upregulation of IGF-1 receptors was observed throughout the spinal cord, significant increases were observed in the cervical and sacral segments compared to controls. IGF-1 receptor immunoreactivity showed a similar pattern to that for 125I-IGF-1 binding, with immunoreactivity being found in the gray matter of the spinal cord and enhanced immunoreactivity occuring in ALS patients compared to controls. In agreement with the distribution of IGF-1 receptors, IGF-1 immunoreactivity was found within the gray matter of the spinal cord. The cartography of IGF-1 receptors in the normal spinal cord as well as the change of these receptors in diseased spinal cord may be of importance in future treatment strategies of ALS.

Double blind cross over trial with deprenyl in amyotrophic lateral sclerosis.

In this paper we present results from a double blind cross over trial with deprenyl, a selective and irreversible monoamine oxidase-B (MAO-B) inhibitor, in 10 patients suffering from amyotrophic lateral sclerosis. The patients were randomised in such a way that half of the patients started with the active drug and half with the placebo treatment. Each patient was given 10 mg deprenyl (eldepryl, 10 mg tablets) per day for 12 weeks and then placebo for the same length of time. There was a drug free period of 12 weeks between the courses. The neurological status of the patients were evaluated every six weeks by using Norris, spinal and bulbar scores and it was observed that all cases deteriorated in their clinical status during the 36 weeks of the controlled study. MAO-B activity in blood platelets was completely inhibited during treatment with deprenyl. In the preliminary analysis performed so far, no obvious retardation in the progress of the disease could be observed with deprenyl treatment.

Monoamine oxidase-B in motor cortex and spinal cord in amyotrophic lateral sclerosis studied by quantitative autoradiography.

The distribution of MAO-B was studied by using an in vitro quantitative autoradiographical method in the post-mortem spinal cord and motor cortex from control and ALS cases. 3H-L-deprenyl was used as a radiotracer. Sections stained with thionine were used to count glial cells. In both control and ALS spinal cords, high density of 3H-L deprenyl binding was observed around the central canal, in the substantia gelatinosa and other grey matter regions. In the ALS cases a pronounced and statistically significant increase of MAO-B was observed in the corticospinal tract, the motor neuron areas and in the ventral white matter. An increase in the number of glial cells in spinal cords from ALS cases was also evident. Moreover, the concentration of MAO-B was highly correlated with glial cell counts in thionine stained sections in various regions of the spinal cord, both in controls and ALS cases. An elevated level of 3H-L-deprenyl binding, in ALS cases, was observed in all the individual laminae of the pre- and post-central gyri of the cerebral cortex. There was no difference in MAO-B concentration between the two groups in the occipital cortex. A substantial increase in the concentration of MAO-B was observed in the white matter of ALS cases. Reactive gliosis has been shown to be associated with neurodegenerative disorders and experimental lesions in animals. The most likely explanation for the increase of MAO-B in ALS and in other neurodegenerative disorders seems to be that the increase is a consequence of the reactive gliosis associated with these disorders.

Reactive gliosis and monoamine oxidase B.

A double-staining method was applied to cryosections of human spinal cord from patients who died with amyotrophic lateral sclerosis (ALS) and corresponding controls in order to investigate cellular content of monoamine oxidase B (MAO-B). 3H-L-Deprenyl emulsion autoradiography was used in combination with histochemical methods for the detection of astrocytes and monocytes/microglia. In the ALS spinal cords an increased number of astrocytes as well as an increased content of MAO-B in reactive species of astrocytes was demonstrated. No significant 3H-L-deprenyl binding was observed in cells derived from the mesoderm, e.g. monocytes or microglia. Furthermore, a sub-population of reactive astrocytes that contained low levels of MAO-B was observed in spinal sections. These findings were further substantiated by studies performed on primary astrocyte cultures.

Differential increases in catecholamine metabolizing enzymes in amyotrophic lateral sclerosis.

The activity of three catecholamine-metabolizing enzymes, monoamine oxidase type A and type B (MAO-A and MAO-B) as well as catechol-O-methyltransferase (COMT), were estimated in homogenates of human spinal cord using radiometric assays. The enzyme activities were determined in postmortem spinal cord tissue from controls and cases with amyotrophic lateral sclerosis (ALS). The activity of MAO-A was below the limit of detectability in both controls and ALS cases. The activities of MAO-B and COMT were evenly distributed at the various spinal levels. The MAO-B activity was substantially elevated in ALS spinal homogenates, whereas only a slight, but not statistically significant, increase in COMT activity was observed. A significant correlation between COMT and MAO-B activities was observed for controls. However, this covariation was not apparent for the ALS cases. These results suggest that the two enzyme proteins are regulated by more complex mechanisms in the spinal cord in amyotrophic lateral sclerosis than simple general increases caused by elevated astroglial cell numbers. In addition, the MAO-A, MAO-B, and COMT activities were estimated in spinal cords from rats treated with the selective MAO-B inhibitor L-deprenyl, a drug with putative neuroprotective effects in neurodegenerative disorders. After 3 weeks of L-deprenyl treatment (0.25 mg/kg/day, sc), the spinal MAO-A and MAO-B activities were decreased by 50 and 80%, respectively. In contrast, the COMT activity was not altered by L-deprenyl administration.

Monoamine oxidase-B in astrocytes.

In the present report we describe the astrocytic localization and content of monoamine oxidase-B (MAO-B) by means of a 3H-L-deprenyl emulsion autoradiography in primary cultures of rat astrocytes, in cryosectioned astrocytoma surgical specimen, and in cryosections of human spinal cords from patients dying in amyotrophic lateral sclerosis (ALS) and controls. The occurrence of MAO-B enzyme protein depends on the degree of cellular differentiation as demonstrated by studies on astrocytes in primary cultures analyzed at two different stages of maturation. Highly differentiated cells exhibited high relative enzyme concentration whereas glioblasts lacked or showed very low contents of MAO-B enzyme. This was further substantiated by studies performed on human astrocytoma tissue using 3H-L-deprenyl emulsion autoradiography in combination with immunohistochemical detection of glial fibrillary acidic protein (GFAP). Regional increases of MAO-B concentration were found in ALS lumbar sections with quantitative 3H-L-deprenyl autoradiography. On the basis of results obtained from double staining for GFAP and MAO-B, the increase in MAO-B seemed to be due to an increased number of astrocytes as well as an increased content of MAO-B in reactive species of astrocytes. A cell culture model has been used that produces cells with morphology and GFAP-content similar to reactive cells. These astrocytes exhibited high relative content of the MAO-B enzyme protein. In the light of the presented data, taking into account the finding that a subpopulation of reactive cells contained low levels of MAO-B, a heterogeneity among reactive astrocytes was observed.

gamma-Aminobutyrate aminotransferase activity in blood platelets of six species.

1. The kinetic parameters of gamma-aminobutyrate aminotransferase (GABA-T) were studied in washed blood platelets from cat, dog, horse, man, mouse and rat. 2. Wide differences were found in the maximal reaction velocity (Vmax) of GABA-T in blood platelets of the six species. 3. A significant increase in the activity of GABA-T was found in the blood platelets of adult rats as compared to those of young rats. 4. However, there was no significant difference in the activity of platelet GABA-T between male and female rats. 5. The activity of human platelet GABA-T estimated in platelet-rich plasma from healthy male subjects was found to be decreased in the presence of plasma and albumin. 6. The decrease was due to the specific binding of the cofactor pyridoxal phosphate to plasma albumin.

Changes in cholecystokinin receptor binding in rat brain after selective damage of locus coeruleus projections by DSP-4 treatment.

Brain cholecystokinin (CCK)- and noradrenergic activities are two neurochemical systems implicated in anxiety and deficits in novelty-related behaviour. In order to clarify a possible interaction between CCK- and noradrenergic neurotransmission in the brain, DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine], a neurotoxin that selectively destroys noradrenaline-containing nerve terminals originating from the locus coeruleus, was administered to rats IP (10 and 50 mg/kg) seven days before decapitation. Noradrenaline uptake was very markedly reduced in the frontal cortex and hippocampus of the DSP-4 treated animals, whereas the decrease in the hypothalamus was smaller but still statistically significant. Dopamine uptake in the corpus striatum, as well as serotonin uptake in the frontal cortex, hippocampus and hypothalamus, were not influenced by DSP-4 treatment. Concomitantly, CCK receptor binding in certain brain regions was markedly affected. Thus, CCK receptor density was significantly higher in the frontal cortex and hippocampus of DSP-4-treated rats. If desipramine (25 mg/kg) was administered before DSP-4 treatment, the DSP-4-induced changes both in noradrenaline uptake and CCK receptor binding were not present, suggesting that both effects were exerted after uptake of the neurotoxin by the nerve terminals. The time-course of the development of changes in CCK-8 binding paralleled with some lag the development of changes in noradrenaline uptake. These findings demonstrate the denervation of noradrenergic input from the locus coeruleus induces certain alterations in the CCKergic neurotransmission. These alterations are similar to those seen in rats with deficits in response to novel stimuli, and may therefore mediate the neophobic responses observed in animals after lesions of noradrenergic innervation of the forebrain.

Characterisation of dopamine and serotonin uptake inhibitory effects of tetrahydroaminoacridine in rat brain.

The effects of 1,2,3,4-tetrahydro-9-aminoacridine (THA) on uptake rates of radioactive 5-hydroxytryptamine (5-HT) and dopamine were investigated in rat diencephalon and striatal homogenates, respectively. Six and eight microM of THA were needed to inhibit 50% of dopamine and 5-HT uptake rates. Kinetic parameters, Km and Vmax, using six different concentrations of dopamine and 5-HT were estimated in the presence or absence of THA. A significant decrease in Vmax without any change in Km values was observed for both dopamine and 5-HT in the presence of THA. The results show that THA is a non-competitive uptake inhibitor of dopamine and 5-HT in the nerve terminals. The re-uptake blocking effect of THA on dopaminergic and serotonergic neurones, following THA treatment, might lead to increased levels of these monoamines in brains of Alzheimer patients and contribute in the therapeutic effects of the drug.

Monoamine oxidase-B in motor cortex: changes in amyotrophic lateral sclerosis.

The occurrence of monoamine oxidase-B in cerebral cortex and white matter in brains from three patients with the diagnosis of amyotrophic lateral sclerosis and three controls was quantified by means of an autoradiographical method. [3H]L-Deprenyl, an irreversible and selective monoamine oxidase-B inhibitor, was used as ligand and the autoradiographs were analysed by computer-assisted densitometry. In both amyotrophic lateral sclerosis and control cerebral cortex, lamina I showed the highest, laminae II and III intermediate, laminae IV, V and VI the lowest [3H]L-deprenyl binding. White matter showed about one-third of the binding in the cortex. Amyotrophic lateral sclerosis cases showed significantly higher binding of [3H]L-deprenyl in all the cortex laminae of the pre- and postcentral gyri. There was no difference in the binding between the amyotrophic lateral sclerosis cases and the controls in area 7 of the occipital cortex, an area which is relatively spared in amyotrophic lateral sclerosis.

Increased binding of 3H-L-deprenyl in spinal cords from patients with amyotrophic lateral sclerosis as demonstrated by autoradiography.

The present investigation has applied quantitative autoradiography and histochemistry to study the regional distribution of MAO-B and its relation to the number of cells in respective regions. L-deprenyl binds irreversibly and quantitatively to the B-form of monoamine oxidase, MAO, and is an ideal 3H-ligand to measure the MAO-B enzyme protein in tissues by means of in vitro autoradiography. The investigation is performed on spinal sections from five controls and five cases with amyotrophic lateral sclerosis (ALS) on cervical, thoracic and lumbar level. The highest density of 3H-L-deprenyl binding was found around the central canal (lamina X). MAO-B was markedly increased (up to 2.5 times of values in controls) specifically in regions of neurodegeneration e.g. motor neuron laminae and corticospinal tracts. There was a high correlation between glial cell count and 3H-L-deprenyl binding with a relation indicating enhanced MAO-B protein in glial cells within areas of neurodegeneration. In contrast the increased microglial cell number in ALS did not show any correlation with 3H-L-deprenyl binding.

Quantitative localization of human brain monoamine oxidase B by large section autoradiography using L-[3H]deprenyl.

The distribution of monoamine oxidase B (MAO-B) in the human brain was studied by quantitative autoradiography using L-[3H]deprenyl as a ligand. Two postmortem brains from patients without any known neurological diseases were used in this study. Cryosections of 100 microns thickness were taken on tape/paper and transferred to gelatinized glass plates. The sections were incubated with 10 nM L-[3H]deprenyl for 1 h and exposed to a film at 4 degrees C for 4 weeks. The autoradiograms were analyzed by computerized densitometry. High L-[3H]deprenyl binding was observed in caudate nucleus, putamen, cingulate gyrus and insula cortex. Moderate to low binding was seen in globus pallidus, temporal and parietal cortex and in various thalamus nuclei. Occipital cortex showed the lowest binding among the cortex regions and white matter the lowest among all the regions studied. All the regions in case 2 (aged 67) showed higher degree of binding when compared with case 1 (aged 58), which is in agreement with previous results showing an increase in MAO-B activity with age. When the specific binding of L-[3H]deprenyl was plotted against the MAO-B activities estimated biochemically in punches from the same areas, a high positive correlation was found.

Monoamine oxidase B in brains from patients with Alzheimer's disease: a biochemical and autoradiographical study.

In vitro quantitative autoradiography using [3H]L-deprenyl, an irreversible and preferential inhibitor of monoamine oxidase B, was performed to investigate the localization of the enzyme in brains from senile dementia of Alzheimer type and control cases. Brains from three male patients with the clinical diagnosis of senile dementia of Alzheimer type and from three male control patients, without any known clinical history of neurological disorder, were obtained at autopsy. Cryosections of 100 microns thickness were mounted on gelatinized glass plates and dried over desiccant for one week at -20 degrees C. The sections were incubated with 10 nM [3H]L-deprenyl for 1 h and then exposed to film for four weeks. The autoradiographs were analysed by computer-assisted densitometry. Monoamine oxidase-B activities were also estimated in 1% homogenates from 10 different regions, using 10 microM beta-[ethyl-14C]phenylethylamine, in order to study the consonance between the autoradiographical and biochemical techniques. Both [3H]L-deprenyl binding and monoamine oxidase-B activities in senile dementia of Alzheimer type were higher than in the controls in all brain regions studied. The increase was highest in the white matter (about 70%) and in the order of 20-50% in the various gray matter regions. A high correlation coefficient (r approximately 0.9) was obtained between [3H]L-deprenyl binding and monoamine oxidase-B activity, both in the senile dementia of Alzheimer type and in the control brains.

Turnover of monoamine oxidase B (MAO-B) in pig brain by positron emission tomography using 11C-L-deprenyl.

Positron emission tomography (PET) was applied to investigate the rate of turnover of pig brain MAO-B. For this purpose 11C-L-deprenyl was used as an irreversible ligand, which bind stoichiometrically to the enzyme. A tracer dose of 11C-L-deprenyl was injected and PET scans performed to obtain baseline deprenyl binding. A high dose of unlabelled deprenyl was then administered to inhibit the enzyme and tracer doses of 11C-L-deprenyl, with subsequent PET analyses, were given at 0, 2, 7, 21 and 42 days. The half-life for the turnover rate calculated was found to be 6.5 days.

Visualization of brain monoamine oxidase B (MAO-B) in dementia of Alzheimer's type by means of large cryosection autoradiography: a pilot study.

Quantitative autoradiography, using 3H-deprenyl, was applied to study the regional localization of MAO-B in large cryosections from 3 control brains and 3 brains from patients with Alzheimer's disease (AD). High 3H-L-deprenyl binding was found in e.g. basal ganglia, cingulate gyrus, and insula cortex. Temporal cortex, parietal cortex, occipital cortex and various nuclei of the thalamus, showed moderate density, while low binding was observed in e.g. white matter. When the brains from the controls were compared with those with AD, the mean values for 3H-deprenyl binding was higher in the latter group, however, more generalised than previously reported.

Insulin-like growth factor 1 (IGF-1) receptors in the human brain: quantitative autoradiographic localization.

The distribution of Insulin-like growth factor 1 (IGF-1) receptors in large cryosections of human brain hemispheres (80-microns) was studied by quantitative autoradiography using 125I-IGF-1 as ligand. Postmortem tissue only from individuals free from neurological diseases was used. The highest densities of IGF-1 receptors were found in the hippocampus, amygdala and parahippocampal gyrus. Intermediate densities were observed in the cerebellum, cerebral cortex and caudate nucleus, whereas low densities of IGF-1 receptors were obtained in the substantia nigra, red nucleus, white matter and cerebral pedunculus. The cartography of IGF-1 receptors in the normal human brain will hopefully be of use in the study of the alteration of these receptors in diseased brain.

Tetrahydroaminoacridine inhibits human and rat brain monoamine oxidase.

The inhibitory effects of 1,2,3,4-tetrahydro-9-aminoacridine (THA) on monoamine oxidase (MAO; EC 1.4.3.4) enzyme activities in human hippocampal and rat striatal homogenates have been studied. The activities of MAO-A and MAO-B were estimated radiochemically, in-vitro, in human hippocampus and rat striatum in the presence of various concentrations of THA with [2-14C]hydroxytryptamine binoxalate (100 microM) and beta-[ethyl-14C]phenylethylamine hydrochloride (20 microM) as substrates for the respective enzyme form. THA was found to inhibit both MAO-A and MAO-B activities reversibly and competitively, with inhibition constants (Ki) of 12.5 microM and greater than 500 microM respectively, of the rat striatal enzymes. From this it can be extrapolated that at therapeutic tissue concentrations of THA (10(-8) to 10(-6) M), more than 20% of the MAO-A activity should be inhibited. Thus it is possible that inhibition of MAO may be involved in the therapeutic action of THA in Alzheimer's disease.

Quantitative autoradiography of nicotinic receptors in large cryosections of human brain hemispheres.

In vitro quantitative autoradiography was used to visualize nicotinic receptors in large cryosections of human brain hemispheres. Sections 80 microns thick of human brain hemispheres were incubated with (-)-[3H]nicotine and the distribution of nicotinic receptors in human brain was studied. Increasing numbers of nicotinic binding sites were observed in the: hippocampus less than cortex less than cerebellum less than substantia nigra less than putamen less than periaqueductal gray. The cartography of nicotinic receptors in the normal human brain will hopefully be of use in the study of the alteration of these receptors in diseased brain.