S. S. Jossan

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.

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.

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.

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.

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 comparing125I-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 for125I-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.

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 Alzheimers disease.

Large-section cryomicrotomy for in vitro receptor autoradiography

A method for processing human postmortem material for application of autoradiography to large cryosections is described. Whole organ or tissue slices 5 mm thick are frozen between copper plates cooled with liquid nitrogen. They are subsequently embedded in carboxymethyl-cellulose, and 40- to 80-microns sections are taken up on adhesive tape and paper and transferred to gelatinized glass plates. Two procedures for incubation of large sections are described. The method is exemplified by autoradiographs of human brain hemisphere ([ 3H]quinuclidinylbenzilate) and whole biceps muscle ([ 3H]alpha-bungarotoxin).

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.

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

SummaryBrain 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 CCK-ergic 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.

Increased binding of3H-L-deprenyI 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 ideal3H-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 of3H-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 and3H-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 with3H-L-deprenyl binding.