Kenneth J. Kellar

Nicotinic acetylcholine binding sites in Alzheimer's disease

In Alzheimers disease (AD), there is a loss of presynaptic cholinergic markers in the cerebral cortex, but the nature of cholinergic receptor changes is unclear. In this study, [3H]acetylcholine and [3H]nicotine were used to label nicotinic cholinergic binding sites in cerebral cortical tissues obtained at autopsy from patients with AD and from matched controls. A consistent and severe loss of nicotinic receptors was found in AD.

In Vivo Regulation of [3H]Acetylcholine Recognition Sites in Brain by Nicotinic Cholinergic Drugs

Abstract: The in vivo regulation of [3H]acetylcholine ([3H]ACh) recognition sites on nicotinic receptors in rat ‐ brain was examined by administering drugs that increase stimulation of nicotinic cholinergic receptors, either directly or indirectly. After 10 days of treatment with the cholinesterase inhibitor diisopropyl fluorophosphate, [3H]ACh binding in the cortex, thalamus, striatum, and hypothalamus was decreased. Scatchard analyses indicated that the decrease in binding in the cortex was due to a reduction in the apparent density of [3H]ACh recognition sites. In contrast, after repeated administration of nicotine (5–21 days), the number of [3H]ACh recognition sites was increased in the cortex, thalamus, striatum, and hypothalamus. Similar effects were observed in the cortex and thalamus following repeated administration of the nicotinic agonist cytisin. The nicotinic antagonists mecamylamine and dihydro‐β‐erythroidine did not alter [3H]ACh binding following 10–14 days of administration. Further, concurrent treatment with these antagonists and nicotine did not prevent the nicotine‐induced increase in these binding sites. The data indicate that [3H]ACh recognition sites on nicotinic receptors are subject to up‐ and down‐regulation, and that repeated administration of nicotine results in a signal for up‐regulation, probably through protracted desensitization at the recognition site.

Differential effects of electroconvulsive shock and antidepressant drugs on serotonin-2 receptors in rat brain.

The effects of chronic administration of electroconvulsive shock and antidepressant drugs on rat brain serotonin-2 (5-HT2) receptors were investigated. Electroconvulsive shock increases the density and antidepressant drugs decrease the density of 5-HT2 receptors labelled by [3H]spiperone.

Measuring nicotinic receptors with characteristics of α4β2, α3β2 and α3β4 subtypes in rat tissues by autoradiography

Comparison of [125I]epibatidine and 5‐[125I]iodo‐3‐(2‐azetidinylmethoxy)pyridine ([125I]A‐85380) autoradiography showed evidence for nicotinic receptor heterogeneity. To identify the receptor subtypes, we performed [125I]epibatidine autoradiography in the presence of cytisine or A‐85380. By comparing these results with binding data from human embryonic kidney (HEK) 293 cells stably transfected with different combinations of rat nicotinic receptor subunits, we were able to quantify three distinct populations of [125I]epibatidine binding sites with characteristics of α4β2, α3β2 and α3β4 receptors. Although the predominant subtype in rat brain was α4β2, non‐α4β2 binding sites were prominent in many regions. In the habenulo‐peduncular system, cerebellum, substantia gelatinosa, and many medullary nuclei, α3β4‐like binding accounted for more than 40% of [125I]epibatidine binding, and nearly all binding in superior cervical ganglion and pineal gland. Other regions enriched in α3β4‐like binding included locus ceruleus, dorsal tegmentum, subiculum and anteroventral thalamic nucleus. Regions enriched in α3β2‐like binding included the habenulo‐peduncular system, many visual system structures, certain geniculate nuclei, and dopaminergic regions. The combination of autoradiography using a broad spectrum radioligand in the presence of selectivecompetitors, and data from binding to defined receptor subtypes in expression systems, allowed us to quantify the relative populations of these three subtypes.

Exposure to nicotine enhances the behavioral stimulant effect of nicotine and increases binding of [3H] acetylcholine to nicotinic receptors

Rats were given daily injections of nicotine sulfate in doses ranging from 0.1 to 0.4 mg/kg. The behavioral effect of these injections was measured as locomotor activity in photocell cages. Repeated administration of the same dose to each rat resulted in an enhancement of the stimulant effect of nicotine. This enhanced behavioral effect was quite pronounced within 5 days of repeated injection. Tissue from the cerebral cortex of these rats, exposed to nicotine for 5 days, was assayed for binding of [3H]acetylcholine to nicotinic receptors. These relatively small doses of nicotine resulted in 18-26% increases in cortical nicotinic receptors, compared to saline-treated rats. Rats exposed to 0.2 mg/kg of nicotine for 5 days and then given saline for 7 days still showed an enhanced behavioral response to nicotine on the eighth day after exposure, and nicotinic binding in the cortex was still elevated. However, 21 days after exposure to nicotine both the behavioral response to nicotine and the binding values had returned to the same values as those of saline-treated rats. These data imply that increased binding of [3H]acetylcholine to nicotinic sites and the enhanced behavioral effect of nicotine are functionally linked.

Reductions in [3H]nicotinic acetylcholine binding in Alzheimer's disease and Parkinson's disease An autobiographic study

In Alzheimers disease (AD) and Parkinsons disease (PD), dysfunction in the basal forebrain cholinergic system is accompanied by a consistent loss of presynaptic cholinergic markers in cortex, but changes in cholinergic receptor binding sites are poorly understood. In the present study, we used receptor autoradiography to map the distribution of nicotinic [3H]acetylcholine binding sites in cortices of individuals with AD and PD and matched control subjects. In both diseases, a profound loss of nicotinic receptors occurs in all cortical layers, particularly the deepest layers.

Chronic nicotine and locomotor activity: influences of exposure dose and test dose

Repeated exposure to nicotine increases both the number of central nicotinic receptors and the behavioral stimulant effect of nicotine. In the present experiments, the behavioral response to nicotine was examined in photocell activity cages. Groups of rats were tested using doses from 0.1 to 1.6 mg/kg both before and after all rats were exposed for 5 days to a common dose of 0.2 mg/kg/day. Prior to the 5-day exposure, there was a dose-related stimulant response to nicotine, with a maximum response seen at 0.4 mg/kg. After the 5-day exposure, the dose-effect curve was shifted upward, so that greater stimulation was produced at each test dose of nicotine.Other groups of rats were exposed for 5 days to doses of nicotine ranging from 0.01 to 0.30 mg/kg/day. On the 6th day all rats received a common test dose of 0.2 mg/kg and their response was measured in the activity cages. In animals exposed to 0.01 mg/kg/day, the test day response was not different from saline controls, but the groups exposed to higher doses showed increased stimulation in response to the common test dose. Measurements of nicotinic receptor binding using [3H]-acetylcholine found increased binding in groups receiving 0.03 mg/kg/day or more, but not in the group that received 0.01 mg/kg/day.The correspondence between the doses that increase behavioral stimulant reactions to nicotine and the doses that increase nicotinic binding suggest that increased receptor numbers may be responsible for the increased behavioral stimulation. However, rats given high doses (1.6 mg/kg, twice per day) did not show increased behavioral stimulation to a test dose of 0.2 mg/kg. In those rats, receptor binding was increased even more than in rats exposed to lower chronic doses. Several hypotheses are offered for this apparent discrepancy.

Muscarinic and nicotinic cholinergic binding sites in Alzheimer's disease cerebral cortex.

The total population of muscarinic receptors and a subpopulation of muscarinic receptors with high affinity for agonists were measured with [3H]quinuclidinyl benzilate and [3H]acetylcholine, respectively, in homogenates of cerebral cortex from control and Alzheimers disease brains. No significant differences between control and Alzheimers diseased cortex were found in either the total population of receptors or the subpopulation with high affinity for agonists in either the frontal or temporal poles. Nicotinic cholinergic receptors labeled by [3H]acetylcholine were measured in homogenates and by autoradiography in the same brain areas. In contrast to muscarinic sites, binding to nicotinic sites was markedly decreased in Alzheimers disease cortex. Autoradiography of [3H]acetylcholine binding to nicotinic sites indicated that in control cortex these sites are more concentrated in laminae IV-VI than in the superficial laminae, and that in Alzheimers disease there is loss of these sites in all cortical laminae.

Differential Regulation of Neuronal Nicotinic Receptor Binding Sites Following Chronic Nicotine Administration

Abstract: Chronic nicotine administration to rats produces an increase in neuronal nicotinic receptors in the CNS. Moreover, the up‐regulated sites labeled by [3H]cytisine in cerebral cortex appear to be composed exclusively of α4 and β2 subunits. It is unknown whether receptor subtypes that do not bind [3H]‐cytisine with high affinity are also affected. In the present studies, we tested the hypothesis that nicotine treatment differentially alters the density of neuronal nicotinic receptor subtypes in rat nervous tissues. Thus, we compared the binding of [3H]cytisine with that of [3H]epibatidine to nicotinic receptors in brain, spinal cord, and adrenal gland from rats that had been injected twice daily with nicotine or saline vehicle for 10 days. Chronic nicotine treatment led to an increase in nicotinic receptor binding sites in the cerebral cortex and in the dorsal lumbar spinal cord, but not in the thalamus. It is important that virtually all of the observed increases could be accounted for by a selective effect on the fraction of receptors exhibiting high affinity for both [3H]‐cytisine and [3H]epibatidine. In contrast, no change in [3H]‐epibatidine binding was seen in the adrenal gland, a tissue that does not exhibit high‐affinity [3H]cytisine binding. These data indicate that, under the conditions used here, nicotine up‐regulates the α4β2 nicotinic receptor subtype, which can be labeled by [3H]cytisine and [3H]epibatidine, but not non‐α4β2 subtypes, which can be labeled by [3H]epibatidine.

Sazetidine-A, A Novel Ligand That Desensitizes α4β2 Nicotinic Acetylcholine Receptors without Activating Them

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels found throughout the central and peripheral nervous systems. They are crucial to normal physiology and have been clearly implicated in nicotine addiction. In addition, they are possible therapeutic targets in a wide range of pathological conditions, including cognitive disorders, Parkinsons disease, and neuropathic pain. Nicotinic ligands are usually classified as agonists (or partial agonists), competitive antagonists, or noncompetitive antagonists. Sazetidine-A is a new nicotinic ligand that shows a different pharmacological profile from any of these known classes of ligands. Sazetidine-A competes with very high binding affinity (Ki ≈ 0.5 nM) and selectivity for the α4β2 nAChR subtype (Ki ratio α3β4/α4β2 ∼ 24,000). Despite its high affinity, sazetidine-A neither activates nAChR channel function nor prevents channel activation when it is applied simultaneously with nicotine. However, when it is pre-incubated for 10 min with the receptors, it potently blocks nicotine-stimulated α4β2 nAChR function (IC50 ≈ 30 nM). The action of sazetidine-A may be explained by its very low affinity for the resting conformation of the α4β2 nAChRs, and its very high affinity for the desensitized state of the receptor. We propose that sazetidine-A is a “silent desensitizer” of nAChRs, meaning that it desensitizes the receptor without first activating it. Furthermore, comparison of the effects of sazetidine-A and nicotine at α4β2 nAChRs suggests that the predominant effects of nicotine and other nicotinic agonists are related to desensitization of the receptors and that sazetidine-A potently mimics these effects.