Jan O. Marcusson

Transmitter deficits in Alzheimer's disease

The pattern of neurotransmitter pathway losses in Alzheimers disease are reviewed. Deficits of the cholinergic pathway from the nucleus basalis, the noradrenergic pathway from the locus coeruleus and the serotoninergic pathway from the raphe nuclei are established. Cortical somatostatin interneurons are affected and dopaminergic neurons may be affected although these may be late or secondary phenomena in the disease process. Other neuronal systems, particularly in the hippocampus and temporal cortex, are also damaged. However, the disease is not one of generalised neuronal atrophy since some neurons are selectively spared. The established pathway-specific losses are discussed in relation to the clinical symptomatology and the pathology of the disorder. The biochemical and histological findings are compared with similar measurements made on tissues from other dementing disorders in an attempt to trace features common to dementias. Finally, as an addendum, a hypothesis is briefly outlined which attempts to explain the common features of the affected neurons and the pathogenesis of the disorder.

The effect of age on the activity and molecular properties of human brain monoamine oxidase

The effect of age upon monoamine oxidase -A and -B (MAO-A and -B) in 23 different regions of human brain was determined. There was a significant positive correlation with age in 19 out of 23 regions for MAO-B, but no positive correlation with age was found for MAO-A. The increased MAO-B activity was found, in 5 out of 5 regions tested, to be due entirely to an increased enzyme concentration, rather than due to an increased molecular turnover number of the enzyme. The responses of the mitochondrial marker enzymes succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) were studied in 5 brain regions, and no consistent change in activity found with age. The lysosomal enzyme acid phosphatase was found to tend towards an increased activity with age. No difference in either the specific activities or molecular characteristics of MAO were found between men and women. Cross-correlation studies of the data, after compensation for the effects of age, indicated that the activities of the two enzyme forms are under some form of organized control across the whole brain. Such a finding is consistent with a genetic regulation of the enzyme forms.

Age‐Correlated Loss of Dopaminergic Binding Sites in Human Basal Ganglia

Abstract: Human caudate nucleus, putamen, substantia nigra, and nucleus accumbens were analyzed for the effects of age on dopaminergic binding sites. Decreases in the number of dopaminergic binding sites were detected with age in caudate nucleus (44 specimens from three sample groups) and substantia nigra (n = 12). In caudate nucleus, the decline in [3H]2‐amino‐6,7‐dehydroxy‐1,2,3,4‐tetrahydronaphthalene sites was three times greater than for [3H]spiperone, but age changes were significant in only two of the three sampling groups. No age changes in binding were detected in the putamen (n = 44) or nucleus accumbens. Age, sex, and tissue source all significantly contributed to variance. However, cause of death, time from death to tissue freezing, and length of storage did not influence dopaminergic binding in the caudate nucleus or putamen. Relative to the life‐span, the age‐correlated decrease in dopaminergic binding sites of human brain approximates that in aging rodent striatum. Comparisons of altered dopaminergic binding with other age‐correlated changes suggest that neuronal loss may not be involved in the loss of binding sites before midlife.

High affinity [3H]paroxetine binding to serotonin uptake sites in human brain tissue

[3H]Paroxetine binding to human brain tissue was characterized. Competition studies in the putamen and frontal cortex revealed single-site binding models for binding sensitive to 5-hydroxytryptamine (5-HT) (Ki 1-3 microM) and citalopram (Ki 0.6 nM), which displaced the same amount of binding. However, desipramine, norzimeldine and fluoxetine displaced additional binding (10-20%) and these competitors fitted two-site binding models with high affinity components in the nanomolar range and low affinity components in the micromolar range. The high affinity components approximated the 5-HT- and citalopram-sensitive binding fraction. Most of the [3H]paroxetine binding sites were protease-sensitive, but the low-affinity (microM) sites appeared to be protease-resistant. Based on these findings, only the [3H]paroxetine binding representing the fraction sensitive to 30 microM 5-HT (or e.g. 0.3 microM norzimeldine), was regarded as specific binding. This binding fraction was saturable with an apparent binding affinity (Kd) of 0.03-0.05 nM throughout the brain. The highest binding densities were obtained in the hypothalamus and substantia nigra (Bmax 500 fmol/mg protein). The basal ganglia reached intermediate densities (Bmax 200 fmol/mg protein), whereas cortical areas had low Bmax values (less than 100 fmol/mg protein). The lowest B max value was noted in cerebellar cortex (30 fmol/mg protein). The [3H]paroxetine binding was competitively inhibited by low concentrations of 5-HT, imipramine and norzimeldine, suggesting that the substrate recognition site for 5-HT uptake was labeled. Compounds active at dopaminergic, noradrenergic, histaminergic, 5-HT1, 5-HT2 and cholinergic muscarinic sites did not affect the binding at 100 microM concentrations. It is concluded that [3H]paroxetine is a marker for the 5-HT uptake site in the human brain, provided that an adequate pharmacological definition of specific binding is performed.

Characterization of [3H]Paroxetine Binding in Rat Brain

Abstract: The binding of the 5‐hydroxytryptamine (5‐HT, serotonin) uptake inhibitor [3H]paroxetine to rat cortical homogenates has been characterized. The effect of tissue concentration was examined and, with 0.75 mg wet weight tissue/ml in a total volume of 1,600 μl, the binding was optimized with an apparent dissociation constant (KD) of 0.03–0.05 nM. Competition experiments with 5‐HT, citalopram, norzimeldine, and desipramine revealed a high (90%) proportion of displaceable binding that fitted a single‐site binding model. Fluoxetine and imipramine revealed, in addition to a high‐affinity (nanomolar) site, also a low‐affinity (micromolar) site representing approximately 10% of the displaceable binding. The specificity of the [3H]paroxetine binding was emphasized by the fact that 5‐HT was the only active neurotransmitter bound and that the serotonin S1 and S2 antagonist methysergide was without effect on the binding. Both 5‐HT‐ and fluoxetine‐sensitive [3H]paroxetine binding was completely abolished after protease treatment, suggesting that the binding site is of protein nature. Saturation studies with 5‐HT (100 μM) sensitive [3H]paroxetine binding were also consistent with a single‐site binding model, and the binding was competitively inhibited by 5‐HT and imipramine. The number of binding sites (Bmax) for 5‐HT‐sensitive [3H]paroxetine and [3H]imipramine binding was the same, indicating that the radioligands bind to the same sites. Lesion experiments with p‐chloroamphetamine resulted in a binding in frontal and parietal cortices becoming undetectable and a >60% reduction in the striatum and hypothalamus, indicating a selective localization on 5‐HT terminals. Together these findings suggest that [3H]paroxetine specifically and selectively labels the substrate recognition site for 5‐HT uptake in rat brain.

Serotonin-2 binding sites in human frontal cortex and hippocampus. Selective loss of S-2A sites with age

Serotonin (S-2) binding sites were characterized in human frontal cortex and hippocampus throughout the lifespan. We found that the S-2 binding sites (labeled by [3H]spiperone and displaced by ketanserin) consisted of two subtypes (S-2A and S-2B) which are discriminated by competition with methysergide. The total S-2 sites in frontal cortex had a Bmax of 360 fmol/mg protein, which is approximately twice that of the hippocampus (160 fmol/mg protein). The density of total S-2 sites decreased with age in the frontal cortex of normal adults (17-100 years, n = 24). The receptor loss was primarily in the S-2A subclass. This loss of S-2A sites occurred primarily after 60 years and decreased to 50% of young adult values by the 10th decade. In the hippocampus, S-2A binding sites decreased with age, but no effects on total S-2 or S-2B sites were detected. A study of infant brains suggested the S-2A subtype in frontal cortex increases postnatally. However, in the infant hippocampus the S-2 binding approximated adult levels.

Titration of human brain monoamine oxidase-A and-B by clorgyline andl-deprenil

SummaryThe interaction of clorgyline andl-deprenil with the-A and-B forms of human brain monoamine oxidase (MAO) has been studied. Both compounds inhibit cerebrocortical MAO in a manner consistent with a ‘suicide’ inactivation of the enzyme. The interaction of clorgyline with the-A form of the enzyme appears to take place almost entirely at specific binding sites, and the conditions required for this inhibitor to ‘titrate’ the concentrations of MAO-A have been elucidated.l-Deprenil has also been used to titrate the concentration of the-B form of MAO in cerebrocortical homogenates, but there is a considerable degree of non-specific binding of this compound. The two inhibitors have been used to titrate the concentrations of the two enzyme forms in frontal cortex homogenates from different age groups. There was a significantly higher MAO-B activity for the age range 73–95 years than for the age range 2–63 years. No significant differences between the two age groups were found for MAO-A. The activity of MAO-A in the samples correlated very well with the concentration of this enzyme form. Titration of the B-form of the enzyme withl-deprenil indicated an increased enzyme concentration with age, although other factors, such as the non-specific binding of this compound, could contribute to this effect.

Specific binding of [3H]imipramine to protease-sensitive and protease-resistant sites.

Abstract: A number of 5‐hydroxytryptamine (5‐HT) uptake inhibitors have been shown to displace the binding of [3H)imipramine to rat cortical membranes in a complex manner with Hill slopes less than unity. Norzimeldine displaced the binding of [3H]imipramine in a biphasic manner with IC50 values for the two components of about 30 nM and 30 μM. This latter site alone was found in tissues that had been treated with a protease. Binding to both of these sites was displaced by 10μM desipramine. The protease‐sensitive [3H]imipramine binding sites were found to be saturable, high‐affinity binding sites with a KD of 8 nM. The number of these sites varied between brain regions and was positively correlated with the regional distribution of [14C]5‐HT but not [3H]noradrenaline uptake. This was not the case however for the protease resistant but desipramine‐displaceable binding sites. Since most previous [3H]imipramine binding studies have been performed with high concentrations of desipramine (10 μ. M) to define “specific binding,” these data would suggest that either protease‐sensitivity or displaceability by 1 μM norzimeldine would give more reliable estimates of the specific binding.

Binding of some antidepressants to the 5-hydroxytryptamine transporter in brain and platelets.

Antidepressant agents with properties to inhibit 5-hydroxytryptamine (5-HT, serotonin) uptake in brain tissue and platelets bind with high affinities to neuronal and platelet membranes. [3H]Imipramine, [3H]paroxetine and [3H]citalopram label specific binding sites related to the 5-HT transporter. [3H]Paroxetine and [3H]citalopram appear to be better ligands than [3H]imipramine. The former label a homogenous population of binding sites, whereas the displaceable binding of [3H]imipramine is heterogenous. Recent observations in several laboratories, which have taken the heterogeneity of [3H]imipramine binding into account, indicate that the binding of antidepressants to the 5-HT transporter probably occurs to the same site that binds 5-HT for transport and not to a separate site as previously suggested. Additional bonds to subsites in close vicinity to the 5-HT recognition site may contribute to the binding. No convincing evidence has been presented of the existence of an endogenous ligand other than 5-HT itself that binds to the [3H]imipramine binding site. Recent studies also suggest that repeated treatment of rats with antidepressant agents does not produce any alterations of the binding of [3H]imipramine or [3H]paroxetine to membranes of cerebral cortex. It is also doubtful whether the density of the 5-HT uptake site in platelets measured with these ligands is decreased in affective disorders as first reported.

Effect of Age on Human Brain Serotonin (S-1) Binding Sites

The effect of age on the binding of [3H]5‐hydroxytryptamine ([3H]5‐HT, serotonin) to postmortem human frontal cortex, hippocampus, and putamen from individuals between the ages of 19 and 100 years was studied. One high‐affinity binding site was observed in adult brains, with a mean KD of 3.7 nM and 3.2 nM for frontal cortex and hippocampus, respectively, and 9.2 nM for putamen. Decreased binding capacities (Bmax) with age were detected in frontal cortex and hippocampus. In putamen a decrease in affinity was noted. Postmortem storage did not significantly contribute to the age‐related changes. No significant sex differences were detected. [3H]5‐HT binding was also studied in brains from human neonates. The specific binding was 1.5–3 times lower than in adult frontal cortex and putamen, and Scatchard analysis suggested more than one binding site. In infant hippocampus a single binding site was observed and except for a premature individual, the binding capacity approximated adult values.