Induction of Fos-immunostaining by nicotine and nicotinic receptor antagonists in rat brain
Introduction
The critical involvement of nicotine in maintaining the use of tobacco products emphasises the importance to study the basis of its effects at the cellular level. The central effects of nicotine involve complex modulation of neuronal firing, neurotransmitter release and neuroendocrine functions. These effects are mediated via neuronal nicotinic acetylcholine receptors (nAChRs) that are ligand-gated ion channels existing in multiple pentameric subunit combinations (see McGehee and Role, 1995 for a review). Until recently, lack of selective ligands able to discriminate between the different subtypes of nAChRs has made it difficult to study functional differences between these receptors. Furthermore, nicotine's pharmacological profile is further complicated by the fact that it may either stimulate or desensitize nAChRs and consequently the stimulatory or inhibitory nicotinic responses converge to produce varying effects in different brain areas (Dani and Heinemann, 1996, Chiodini et al., 1999).
Induction of immediate-early genes (IEGs) has been extensively studied as a marker for postsynaptic activation of cells in the central nervous system (Sagar et al., 1988). Furthermore, brain IEG expression has been successfully used to map neuronal pathways activated by nicotine. Administration of nicotine induces the expression of the prototypical immediate early gene c-fos and its protein product Fos in restricted brain regions of rats including target areas of the midbrain dopamine neurones, areas of the hypothalamus and areas related to the optic system (Ren and Sagar, 1992, Matta et al., 1993, Pang et al., 1993, Kiba and Jayaraman, 1994, Salminen et al., 1996, Salminen et al., 1999, Salminen et al., 2000). Interactions with dopaminergic and glutamatergic systems have been demonstrated in the dopamine-innervated areas where antagonists of both dopamine D1 and N-methyl-D-aspartate (NMDA) preferring glutamate receptors inhibited the nicotine-induced increases of Fos-like immunostaining (Fos IS; Kiba and Jayaraman, 1994, Nisell et al., 1997, Schilström et al., 2000a).
The present experiments were designed to study the ability of different nAChR antagonists to block nicotine-induced Fos responses in various regions of rat brain. We also studied the extent to which these antagonists affect brain IEG expression by themselves, because in the course of the experiment we discovered that all three antagonists elevated Fos IS in several of the brain areas studied. Three different nAChR antagonists included in the study were dihydro-beta-erythroidine (DHE, Williams and Robinson, 1984), the competitive antagonist to the nicotine binding site, methyllycaconitine (MLA; Ward et al., 1990), the α7 nAChR-selective antagonist, and mecamylamine, the ion channel blocking agent. The nAChR antagonists used in the present study differ in their abilities to inhibit nicotine's behavioural actions in rats and mice, presumably due to their different affinities to different subtypes of nAChRs (Stolerman et al., 1997, Damaj et al., 1999, Blondel et al., 2000). All three antagonists have been suggested to reduce the rewarding effects of nicotine in rats (Huston-Lyons and Kornetsky, 1992, Epping-Jordan et al., 1998, Panagis et al., 2000). Mecamylamine has previously been shown to block or reduce nicotine-induced Fos expression in several brain areas (Ren and Sagar, 1992, Pang et al., 1993, Kiba and Jayaraman, 1994, Schilström et al., 2000a). In a recent report blockade of nicotine-induced Fos IS by MLA in the nucleus accumbens was described (Schilström et al., 1999). To our knowledge, the effect of DHE either alone or in combination with nicotine on Fos IS has not been previously studied. A preliminary report of our results has been previously published (Seppä et al., 1999).
Section snippets
Animals
Male Wistar rats weighing 200–250 g were used in the experiments. They were kept under a 12 h light/dark cycle (lights on at 0600 h) at an ambient temperature of 20–23°C. Rat chow and tap water were available ad libitum. The rats were habituated to handling and restraint for twice daily for 10 min periods on two successive days before the experiment. During the habituation and throughout the experiments the rats were housed singly to a cage. The experimental procedure was approved by the local
Fos IS in rats treated with saline
In control rats given saline i.p. and s.c., the number of Fos positive nuclei varied largely depending on the brain region studied (Fig. 1, Fig. 2, Fig. 3, Fig. 4, left columns). Moderate immunoreactivity was detected in all areas containing dopamine (mPFC, Cg, NAcc, CPu and CeA) and in the PVN. Furthermore, Fos IS was detectable also in the Par, SON and SuG. The MT and the IPN were devoid of Fos-positive nuclei in saline-treated rats.
The effect of acute nicotine on Fos IS
In saline-pretreated rats given nicotine, Fos IS was
Discussion
In accordance with earlier studies, we found that acute nicotine administration significantly increased Fos IS in ten out of the eleven regions investigated, excluding the mPFC (Ren and Sagar, 1992, Matta et al., 1993, Pang et al., 1993, Kiba and Jayaraman, 1994, Salminen et al., 1996, Panagis et al., 1996, Nisell et al., 1997, Schilström et al., 2000a; but see however Mathieu-Kia et al., 1998). In cortical, limbic and hypothalamic areas nicotine increased the number of Fos positive cells by
Acknowledgments
The authors wish to thank Ms Marika Harjunen and Ms Reija Salminen for excellent technical assistance. This work was carried out with support from the Faculty of Science, University of Helsinki to T.S. and from the Finnish Cultural Foundation to O.S.
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2012, Brain Research BulletinCitation Excerpt :Following an appropriate external or internal stimulus, the immediate early gene c-fos expression leads to the synthesis of the c-Fos protein in the cytoplasm and then the protein immediately translocates in the nucleus and by binding to specific sites on the genes, it triggers the genomic expression. For example, vasopressin and oxytocin neurons respond to stress [18,23,47], hypovolemia [13,42], hyperosmosis [10,14,15,33,40], and hemorrhage [36], as well as different types of neurotransmitter agonists including nicotine [32,34] by expressing c-Fos protein. It has been shown that nicotine [28] or carbachol [43] injections can increase c-Fos immunoreactivity in different parts of the brain including hypothalamus.