Elsevier

Brain Research

Volume 765, Issue 1, 8 August 1997, Pages 135-140
Brain Research

Research report
Nicotine protects cultured cortical neurons against glutamate-induced cytotoxicity via α7-neuronal receptors and neuronal CNS receptors

https://doi.org/10.1016/S0006-8993(97)00556-8Get rights and content

Abstract

We examined the effects of nicotine on glutamate-induced cytotoxicity using primary cultures of rat cortical neurons. The cell viability decreased significantly when cultures were exposed to glutamate for 10 min and then incubated with glutamate-free medium for 1 h. The exposure of cultures to nicotine (10 μM) for 8–24 h prior to glutamate application ameliorated the glutamate-induced cytotoxicity, with no significant effect of nicotine alone on the cell viability. Neuroprotection by nicotine was dependent on the incubation period. α-bungarotoxin (α-BTX) and methyllycaconitine (MLA), both of which are α7-neuronal receptor antagonists, and dihydro-β-erythroidine (DHβE), a neuronal central nervous system (CNS) receptor antagonist, each significantly antagonized the protection by nicotine against glutamate-induced cytotoxicity. Ionomycin, a calcium ionophore, and S-nitrosocysteine (SNOC), a nitric oxide (NO) donor, also induced cytotoxicity in a manner similar to glutamate. Nicotine protected cultures against ionomycin-induced cytotoxicity, but not against SNOC-induced cytotoxicity. These results suggest that nicotine protects cultured cortical neurons against glutamate-induced cytotoxicity via α7-neuronal receptors and neuronal CNS receptors by reducing NO-formation triggered by Ca2+ influx.

Introduction

The degeneration of the central cholinergic system is the primary pathological manifestation of cognitive dysfunction. In patients with Alzheimer's disease, there is much evidence that the memory and attention deficits are due to the degeneration of the cholinergic systems that originate in the nucleus basalis of Meynert (NBM) and innervate the neocortex [8]. Although attention has been focused on the drugs that prevent the breakdown of acetylcholine, such as physostigmine and tetrahydroacridine, recent evidence has suggested a role of nicotinic cholinergic receptors and some effects of nicotine and its analogs in cholinergic dysfunction. Nicotine has improved both the performance of cognitively demanding tasks in normal healthy adult human subjects [28], and information processing and attention in patients with Alzheimer's type dementia [22]. Moreover, in animal models of cholinergic disturbance produced by NBM lesions, chronic pretreatment with nicotine reduced the cell loss in the neocortex [23]. These findings suggest that nicotine has not only a therapeutic effect on cholinergic dysfunction, but also cytoprotective effects on cholinergic degeneration.

Cortical neurodegeneration in some neuropathies has been attributable to glutamate [17]. Recent in vitro studies using cultured cortical neurons revealed that brief glutamate exposure produces delayed neurodegeneration 7, 12, in which N-methyl-d-aspartate (NMDA) receptors play crucial roles [19]: NMDA receptor stimulation induces Ca2+ influx into the cells through NMDA receptor channels, triggering nitric oxide (NO) formation; the synthesized NO diffuses to adjacent cells, resulting in glutamate-related cell death [3]. The search for the neuroprotective factor against glutamate neurotoxicity revealed the ability of some neurotransmitter-related agents to rescue the neurons from glutamate-related cell death. However, there is little information concerning the cholinergic protection against the neurotoxicity. We have previously reported that nicotine protected cultured cortical neurons against NMDA receptor-mediated glutamate cytotoxicity [4]. The present study was performed to determine the nicotinic receptor subtypes mediating nicotine-induced neuroprotection in cortical cultures. We also investigated the mechanism of the neuroprotective action of nicotine by examining the effects of nicotine on calcium ionophore-induced and NO donor-induced neurotoxicity, which are likely to be involved in the glutamate-neurotoxicity pathway.

Section snippets

Cell culture

Primary cultures were obtained from the cerebral cortex of fetal rats (17–19 days' gestation). The procedures have been described previously 2, 4, 24, 25. Briefly, single cells dissociated from the whole cerebral cortex of fetal rats were plated on plastic coverslips which were placed in 60-mm Falcon dishes (4.8–5.1×106 cells per dish). Cultures were incubated in Eagle's minimal essential salt medium (Eagle's MEM) supplemented with 10% heat-inactivated fetal bovine serum (1–7 days after

Nicotine protection against glutamate neurotoxicity

The exposure of cultured cortical neurons to glutamate induced marked cell death. Trypan blue exclusion revealed that the cell viability was decreased by 10-min treatment with 1 mM glutamate, whereas most of the cells without drug treatment had non-stained, bright cell bodies (Fig. 1A,B). Nicotine protected the cultures from glutamate neurotoxicity. Treatment with 10 μM nicotine for 24 h prior to glutamate exposure significantly reduced the number of cells stained with Trypan blue (Fig. 1C and

Discussion

In the present study, we demonstrated that: (1) nicotine applied prior to glutamate exposure protected cultured cortical neurons against glutamate neurotoxicity in a time-dependent manner; (2) the protective effect was attenuated by a simultaneous application of each subtype-specific antagonist, (an α7-neuronal receptor antagonist and a neuronal CNS receptor antagonist) with nicotine; and (3) nicotine also prevented ionomycin-induced neurotoxicity, but not that induced by SNOC.

The concentration

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