Cholinomimetics increase glutamate outflow via an action on the corticostriatal pathway: implications for Alzheimer's disease.

Physostigmine, the acetylcholinesterase inhibitor (0.3 mg/kg, i.m.), increased extracellular glutamate but not aspartate concentrations in the striatum of anaesthetised rats, determined using microdialysis and HPLC. The rise was both tetrodotoxin and calcium dependent. In contrast, neither physostigmine (10 microM) added to the perfusion fluid nor vehicle (injected intramuscularly) affected amino acid concentrations. To obtain evidence that the action of acetylcholine was to modulate positively cortical pyramidal neurone activity via the M1 receptor, the selective M1 agonist PD 142505-0028 (10 microM) was topically applied to the frontal cortex. Like physostigmine, PD 142505-0028 rapidly increased glutamate but not aspartate concentrations in the striatum. Moreover, the effect of intramuscular physostigmine was blocked by a topically applied M1 antagonist. These new data add to our hypothesis that cholinomimetics increase pyramidal neurone function.

NMDA-induced glutamate and aspartate release from rat cortical pyramidal neurones: evidence for modulation by a 5-HT1A antagonist.

1. We have investigated an aspect of the regulation of cortical pyramidal neurone activity. Microdialysis was used to assess whether topical application of drugs (in 10 microliter) to fill a burr hole over the frontal cortex, where part of the corticostriatal pathway originates, would change concentrations of the excitatory amino acids glutamate and aspartate in the striatum of the anaesthetized rat. 2. Topical application of N-methyl-D-aspartate (NMDA, 2 and 20 mM) dose-dependently increased glutamate and aspartate concentrations in the striatum. Coapplication of tetrodotoxin (10 microM) blocked the NMDA-evoked rise in these amino acids. A calcium-free medium, perfused through the probe also blocked the rise, indicating that it was due to an exocytotic mechanism in the striatum. 3. It was hypothesized that the rise observed was due to an increase in the activity of the corticostriatal pathway. As 5-hydroxytryptamine1A (5-HT1A) receptors are enriched on cell bodies of corticostriatal neurones, a selective 5-HT1A-antagonist (WAY 100135) was coapplied with the lower dose of NMDA. Compared to NMDA alone, coapplication of 50 microM WAY 100135 significantly increased glutamate release. This effect was sensitive to tetrodotoxin and calcium-dependent. Application of 50 microM WAY 100135 alone significantly enhanced glutamate release above baseline; this was also tested at 100 microM (not significant). 4. Compared to NMDA alone, coapplication of WAY 100135 (20 microM) significantly enhanced aspartate release; the mean value was also increased (not significantly) with 50 microM. This rise was calcium-dependent, but not tetrodotoxin-sensitive. WAY 100135 (100 microM) reduced NMDA-induced aspartate release. WAY 100135 (100 microM) reduced NMDA-induced aspartate release. Application of the drug alone had no effect on basal aspartate release.5. Coapplication of the 5-HT1A agonist, 8-OHDPAT (5 sanM) with NMDA did not affect the NMDA evoked increase in glutamate and aspartate.6. Topical application of high potassium (100 sanM) to the surface of the cortex did not result in a detectable rise in striatal glutamate or aspartate.7. Perfusion of WAY 100135 (tested at 50 microM) through the dialysis probe did not affect glutamate oraspartate concentrations.8. It was concluded that a selective 5-HT1A-antagonist can increase the activity of corticostriatal pyramidal neurones. As in Alzheimer's disease hypoactivity of pyramidal neurones almost certainly exists, a selective 5-HT1A-antagonist may be potentially useful in the treatment of the cognitive symptoms of this disease.

Postmortem brains reveal similar but not identical amyloid precursor protein-like immunoreactivity in Alzheimer compared with other dementias.

Concentrations of amyloid precursor protein (APP)-like immunoreactivity (APPLIR) have been determined by Western blotting in a soluble fraction and two membrane fractions of two areas of brain cortex from patients with Alzheimer's disease (AD) and other dementias. There were no significant differences between AD and other cases in species with the Kunitz protease inhibitor domain. However, the total soluble APPLIR was higher in AD and it was hypothesized that this relates to cholinergic hypoactivity.

Brain membrane serine protease activity in human cortex compared with rat: implication for Alzheimer's disease.

Cerebral cortex from humans and rats was extracted sequentially with detergent-containing and low-ionic-strength buffers. The resulting pellet was extracted with detergent/high-ionic-strength buffer to yield a soluble enzyme preparation. This was incubated with substrate prepared from rat cerebral cortical membranes containing amyloid precursor protein-like immunoreactivity (APPLIR) of 116 kD approximate apparent molecular mass. The effectiveness of various enzyme preparations to degrade APPLIR was: routine-post-mortem (pm)-delay human samples > rat pup > short-pm-delay human samples >> adult rat. In incubations with human samples only a 100-kD product accumulated. The activity in human brain was inhibited by phenylmethylsulphonylfluoride, insensitive to Ca2+, correlated with pyramidal neurone numbers but not those of astrocytes and was not significantly higher in Alzheimer's disease compared with controls. These data are discussed in terms of other approaches for studying proteolytic activity to explain the deposition of beta-amyloid protein in this disease.

beta-Amyloid precursor protein isoforms show correlations with neurones but not with glia of demented subjects.

Post-mortem cerebral cortex from 15 demented patients was specially collected to minimise autolysis and two membrane fractions and one soluble fraction were quantitatively examined for the major species of beta-amyloid precursor protein (APP) of high apparent molecular mass (> or = 80 kDa) together with the major mRNA species encoding APP isoforms. The number of pyramidal neurones and astrocytes, putative biochemical indices of interneurones and pyramidal neurones, and choline acetyl transferase activity were also determined. Multiple regression analysis has been used to investigate intercorrelations of APP species with biochemical and morphometric measures, free of any effects of confounding demographic variables. Subjects with Alzheimer's disease showed a loss of cholinergic activity and D-aspartate uptake compared with patients with other causes of dementia. The major finding of the study is that measures of neurones rather than astrocytes most closely correlate with the concentration of APP. Pyramidal cell numbers were positively correlated with mRNA for APP695. APP in the soluble fraction showed a negative correlation with pyramidal cell numbers and cholinergic activity. These results indicate that neurones within the cerebral cortex are the major source of APP, and that secretion of APP is dependent upon cortical pyramidal neuronal activity and cholinergic activity.

Neurotransmitters and second messengers in aging and Alzheimer's disease.

A substantial loss of cortical cholinergic nerve endings, along with a much more circumscribed cortical degeneration of pyramidal neurons, almost certainly causes glutamatergic hypoactivity in live Alzheimer's patients. These selective pathologies are discussed in terms of therapy. An additional effect of some proposed treatments is emerging as there is evidence that processing pathways for beta-amyloid precursor proteins in cortical pyramidal neurons, a target cell for acetylcholine, are affected by neuronal activity.

Preliminary neurochemical findings in non-Alzheimer dementia due to lobar atrophy.

Non-Alzheimer's dementia due to lobar atrophy had choline acetyltransferase activities comparable with control rather than Alzheimer's disease values, based on 3 autopsy proven cases on Pick's disease and biopsies from 3 examples of dementia of frontal lobe type. Muscarinic cholinergic receptors were relatively spared only in Alzheimer's disease. Serotonin receptors were markedly reduced (based on Pick cases) whereas measures that reflected presynaptic serotonergic activity were either not affected or increased. Cerebrospinal fluid and brain tissue measurements suggested that inhibitory interneurones and dopamine release were relatively spared. There was no in vitro evidence of hypometabolism.

Serotonergic pathology is not widespread in Alzheimer patients without prominent aggressive symptoms.

Behavioural symptoms of Alzheimer's disease, such as aggression, may determine the care patients required. Most postmortem neurochemical studies have been of institutionalized patients and conclusions drawn from these may not be valid for all patients. We have shown that serotonin 2 receptors are not lost from 12 of the 13 areas of cerebral cortex examined in the patients assessed to be free of aggressive symptoms. This has been interpreted as representing the relative preservation of cortical interneurones. In contrast choline acetyltransferase activity was reduced in all areas whereas serotonin content was reduced in only 2 of the 4 areas examined.

Protease 'Clipsin' extract activity reflects demographic characteristics of human brain.

'Clipsin' and other peptide bond hydrolase activities that appear to be integral membrane proteins, including two implicated in the formation of the histological hallmarks of Alzheimer's disease, were assayed in the frontal and parietal cortex from a large (n = 45) series of postmortem human brains. Tissues were extracted sequentially with detergent-containing and low ionic strength buffers. The final extracts obtained with detergent-high ionic strength buffer were assayed for peptide-bond hydrolase activity using radiolabelled casein and four chromophore-linked peptide substrates. Hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and alpha-casein showed evidence of sensitivity to the presence of Alzheimer's disease (n = 22) and some of the seven other demographic features (postmortem delay; mode of death in the case of one substrate) of the brain samples considered. By contrast, activity towards carbobenzoxy(Z)-Leu-Leu-Glu-2-naphthylamide, Z-Val-Lys-Met-4-methyl-coumaryl-7-amide and Z-Val-Lys-Lys-Arg-4-methoxy-2-naphthylamide were independent of all factors. The results are discussed in terms of damage to a sub-population of protease-containing membranes.

5-Hydroxytryptamine1A but not 5-hydroxytryptamine2 receptors are enriched on neocortical pyramidal neurones destroyed by intrastriatal volkensin.

Experimental lesions followed by binding of [3H]8-hydroxy-2-(n-dipropylamino)tetralin (8-OH-DPAT) and [3H]ketanserin to cryostat sections and quantitative autoradiography were used to investigate the cellular localization of 5-hydroxytryptamine1A (5-HT1A) and 5-hydroxytryptamine2 (5-HT2) receptors in the neocortex of the rat. The lesions were produced by intrastriatal injections of either volkensin (2 and 6 ng) or ricin (10 ng): both are suicide transport agents, but only the former is retrogradely transported in the central nervous system. Only animals treated with volkensin showed cortical receptor changes and these were almost exclusively confined to the 5-HT1A site. The binding of [3H]8-OH-DPAT was significantly reduced in rats receiving 2 or 6 ng volkensin in the deeper cortical layers of areas Fr1/Fr2 of neocortex ipsilateral to the striatal lesion. Quantitative histological analysis of adjacent sections had previously revealed a significant loss of large infragranular pyramidal neurones with sparing of both interneurones and supragranular pyramidal neurones. There was no significant reduction in [3H]8-OH-DPAT binding in superficial layers. In cortical areas, Par1/Par2 [3H]8-OH-DPAT binding was significantly reduced (2 and 6 ng animals) in both superficial and deeper cortical layers, but quantitative histological analysis has not been performed. The binding of [3H] ketanserin was unaffected except for the most superficial layers of Par1/Par2, where binding was significantly reduced in only the 2 ng animals. There was no reduction in [3H]8-OH-DPAT binding after intrastriatal ricin injection, which caused as much cell loss in the striatum as 2 ng of volkensin, but did not destroy cortical pyramidal neurones.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterisation of the glycine modulatory site of the N-methyl-D-aspartate receptor-ionophore complex in human brain.

[3H]Glycine binding and glycine modulation of [3H]MK-801 binding have been used to study the glycine allosteric site associated with the N-methyl-D-aspartate receptor complex in postmortem human brain. The effect of glycine on [3H]MK-801 binding appeared sensitive to duration of terminal coma, and possibly postmortem delay. Thirty percent of the binding occurred in a subfraction of brain tissue and did not show enhancement by glycine and glutamic acid. [3H]Glycine binding to a subfraction free from this component was studied and showed high specific binding. KD and Bmax values showed considerable intersubject variability which did not appear to be due to demographic features or to tissue content of amino acids with an affinity for this site. The pharmacological characteristics of binding in this subfraction and a correlation between Bmax values and the maximal enhancement of [3H]MK-801 binding by glycine are consistent with [3H]glycine binding occurring to an N-methyl-D-aspartate receptor complex associated site. Further support for this is provided by a significantly lower Bmax value for [3H]glycine binding in subjects with Alzheimer's disease and reduced glycine enhancement of [3H]MK-801 binding. However, the effect of perimortem factors makes it difficult to confidently attribute this solely to a disease-related change in the receptor. The possible role of the glycine allosteric site in the treatment of neuropsychiatric disorders is discussed.

The N-methyl-D-aspartate receptor complex in Alzheimer's disease: reduced regulation by glycine but not zinc.

The binding of [3H]MK-801 to the N-methyl-D-aspartate receptor complex of well-washed cortical membranes from brains of examples of Alzheimer's disease and controls has been determined in incubations containing either glutamate or glycine plus glutamate. No changes were detected in the IC50 values for inhibition by zinc in the Alzheimer's samples compared to control although 'glycine-dependent' binding of the [3H]-ligand was significantly reduced in Alzheimer's disease.

Reduced glycine stimulation of [3H]MK-801 binding in Alzheimer's disease.

The novel N-methyl-D-aspartate receptor channel ligand (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine maleate ([3H]MK-801) has been utilized to label this receptor in human brain tissue. Characteristics of [3H]MK-801 binding to well-washed membranes from 17 control subjects and 16 patients with Alzheimer's disease were determined in frontal, parietal, and temporal cerebral cortex and cerebellar cortex. In control tissue the pharmacological specificity of the binding of this substance is entirely consistent with the profile previously reported for rat brain. Binding could be stimulated by the addition of glutamic acid to the incubation medium; addition of glycine produced further enhancement which was not prevented by strychnine. The specificity of the effects of these and other amino acids on the binding was the same as in the rat. In Alzheimer's disease significantly less binding was observed in the frontal cortex under glutamate- and glycine-stimulated conditions. This appears to be associated with a reduced affinity of the site whereas the pharmacological specificity of the site remained unchanged. The effect did not appear to be due to differences in mode of death between Alzheimer's disease and control subjects and is unlikely to be related to factors for which the groups were matched. In contrast, binding was not altered in the absence of added amino acids and presence of glutamate alone. These results imply that in the cerebral cortex the agonist site and a site in the cation channel of the receptor are not selectively altered, but that their coupling to a strychnine-insensitive glycine recognition site is impaired.

Loss of glycine-dependent radioligand binding to the N-methyl-D-aspartate-phencyclidine receptor complex in patients with Alzheimer's disease.

Well washed membranes have been prepared from samples of cerebral cortex of control subjects and patients with Alzheimer's disease, obtained both at post mortem and by neurosurgical procedures earlier in the course of the disease. Binding to these membranes of two radioligands for the N-methyl-D-aspartate-phencyclidine receptor complex has been determined in the presence and absence of glycine. Glycine increased the binding in both control and Alzheimer tissue samples. At one concentration of radioligand, in the presence of glycine there was less binding to post-mortem samples, which Scatchard analysis showed was associated with a 36% loss of sites. In rare neurosurgical samples, there was also a loss of binding of radioligand which suggests that the effect is not due to post-mortem artefacts or epiphenomena. These new results may have implications for the symptomatic and preventative treatment of Alzheimer's disease.

Possible neurotransmitter basis of behavioral changes in Alzheimer's disease.

Serotonin, 5-hydroxyindoleacetic acid, and homovanillic acid concentrations have been determined in 10 areas of the cerebral cortex from 17 subjects with Alzheimer's disease and 18 control subjects. The dopamine metabolite was not reduced in any area, whereas both indoleamines were reduced in the superior frontal, inferior temporal, and fusiform gyri, and the temporal pole. These areas and areas of the parietal cortex, where there were no changes in concentration, have not previously been reported on. We argue that the large loss of indoleamines from the frontal lobe (to 50-63% of control values) is rather unexpected based on other biochemical measurements and may relate to behavioral changes.

Topographical distribution of neurochemical changes in Alzheimer's disease.

Biochemical indices of cortical nerve cells affected in Alzheimer's disease have been proposed (excitatory dicarboxylic amino acid, EDAA, sodium-dependent carrier; phosphate-activated glutaminase activity; serotonin type 2 recognition site; somatostatin-like immunoreactivity). These and the content of EDAAs and two related amino acids, and choline acetyltransferase (ChAT) activity have been measured in up to 13 areas of cerebral cortex and the cerebellar cortex from 16 patients with Alzheimer's disease and 17 controls. Reduction of the index of the serotonin recognition site, somatostatin content and another biochemical index of interneurones coincide and indicate a rather unexpected focal loss of such neurones from the parietal lobe. No unequivocal measure of the integrity of pyramidal neurones could be established as the content of no amino acid was reduced, the index of the EDAA carrier showed evidence of change in few brain regions and glutaminase activity was subject to unexplained variability. ChAT activity alone closely paralleled a previous report of the distribution of morphological degeneration. The results are discussed in relation to therapy and positron emission tomography.

Excitatory amino acid-releasing and cholinergic neurones in Alzheimer's disease.

Brains of normal controls and patients with primary degenerative dementia were investigated for choline acetyltransferase (ChAT) activity in the frontal, temporal and parietal cortex, hippocampus, amygdala and thalamus. A few patients with Alzheimer's disease were unusual as the cholinergic marker was unaffected, except in the amygdala. Other patients with dementia and undiagnosed neurodegenerative disorder had elevated cortical ChAT activity. The interpretations offered are: (a) the syndrome of dementia and Alzheimer pathologic change precedes significant loss of cortical cholinergic innervation; (b) denervation in dementia can occur early in olfactory areas, exemplified here by the amygdala; (c) dementia is associated with the loss of non-cholinergic structure. An indication of structures involved was given by loss of a marker of excitatory amino acid-releasing neurones.