Elsevier

Neuropharmacology

Volume 41, Issue 4, September 2001, Pages 486-495
Neuropharmacology

Induction of Fos-immunostaining by nicotine and nicotinic receptor antagonists in rat brain

https://doi.org/10.1016/S0028-3908(01)00093-4Get rights and content

Abstract

Using Fos protein immunohistochemistry, we have studied the effects of acute nicotine (0.5 mg/kg s.c.) and nicotinic acetylcholine receptor (nAChR) antagonists in eleven rat brain areas. Acute nicotine elevated Fos-like immunostaining (Fos IS) significantly in all studied areas except the medial prefrontal cortex. Nicotine increased the Fos IS in cortical, limbic and hypothalamic areas by 2–10-fold, and in the interpeduncular nucleus as well as in the visual areas the increases were 15–150-fold. When given alone, the nAChR antagonists mecamylamine (1.0 or 5.0 mg/kg i.p.) and dihydro-beta-erythroidine (DHE; 1.4 or 2.8 mg/kg i.p.) increased Fos IS in most brain areas maximally by 2–10-fold, but methyllycaconitine (MLA; 4.0 mg/kg i.p.) only in three areas and maximally by 4-fold. The efficacy of nAChR antagonists in blocking nicotine's effects on Fos IS varied noticeably with respect to region and antagonist, and the combined effect of nicotine+antagonist did not exceed that of either treatment alone. Mecamylamine and DHE significantly reduced nicotine-induced Fos IS in most of the studied areas, and MLA only in two areas. Thus, nAChRs seem to mediate the effects of nicotine on Fos IS, and the differences in the effects of the antagonists studied suggest that more than one subtype of nAChRs are involved. The present experiments also provide evidence that nAChR blockade itself may result in increased Fos protein expression in the brain. This could be due to blockade of presynaptic nAChRs modulating transmitter release or interruption of complex polysynaptic feedback pathways.

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.

References (51)

  • O. Salminen et al.

    Expression of Fos protein in various rat brain areas following acute nicotine and diazepam

    Pharmacology Biochemistry and Behavior

    (1996)
  • O. Salminen et al.

    Effect of acute nicotine on Fos protein expression in rat brain during chronic nicotine and its withdrawal

    Pharmacology, Biochemistry and Behavior

    (2000)
  • J.W. Turek et al.

    A sensitive technique for the detection of the α7 neuronal nicotinic acetylcholine receptor antagonist, methyllycaconitine, in rat plasma and brain

    Journal of Neuroscience Methods

    (1995)
  • C. Vidal et al.

    Pharmacological profile of nicotinic acetylcholine receptors in the rat prefrontal cortex: an electrophysiological study in a slice preparation

    Neuroscience

    (1989)
  • J.M. Ward et al.

    Methyllycaconitine: a selective probe for neuronal α-bungarotoxin binding sites

    FEBS Letters

    (1990)
  • S. Wonnacott

    Presynaptic nicotinic ACh receptors

    Trends in Neurosciences

    (1997)
  • A. Blondel et al.

    Characterisation of the effects of nicotine in the five-choice serial reaction time task in rats: antagonist studies

    Psychopharmacology

    (2000)
  • A. Chaudhuri

    Neural activity mapping with inducible transcription factors

    Neuroreport

    (1997)
  • P.B.S. Clarke et al.

    Nicotinic binding sites in rat brain: autoradiographic comparison of [3H]acetylcholine, [3H]nicotine and [3H]-α-bungarotoxin

    Journal of Neuroscience

    (1985)
  • A. Contestabile et al.

    Afferent connections of the interpeduncular nucleus and the topographic organization of the habenulo-interpeduncular pathway: a HRP study in the rat

    Journal of Comparative Neurology

    (1981)
  • M.I. Damaj et al.

    Pharmacological characterization of nicotine-induced seizures in mice

    Journal of Pharmacology and Experimental Therapeutics

    (1999)
  • M.P. Epping-Jordan et al.

    Dramatic decreases in brain reward function during nicotine withdrawal

    Nature

    (1998)
  • S.R. Grady et al.

    Nicotinic acetylcholine receptor mediated acetylcholine release from mouse interpeduncular nucleus synaptosomes

    Society for Neuroscience Abstracts

    (1999)
  • R. Gray et al.

    Hippocampal synaptic transmission enhanced by low concentrations of nicotine

    Nature

    (1996)
  • R.E. Harlan et al.

    Drugs of abuse and immediate-early genes in the forebrain

    Molecular Neurobiology

    (1998)
  • Cited by (26)

    • Effects of chronic nicotine exposure on Δ<sup>9</sup>-tetrahydrocannabinol-induced locomotor activity and neural activation in male and female adolescent and adult rats

      2020, Pharmacology Biochemistry and Behavior
      Citation Excerpt :

      CB1 receptors are widely distributed throughout the brain and thus brain regions of interest in the present study included those which have been found to exhibit increased expression of c-fos in response to acute THC administration, specifically the NAc, dorsal striatum, lateral septum (LS), bed nucleus of the stria terminalis (BNST), hypothalamus, and thalamus (Miyamoto et al., 1996; Erdtmann-Vourliotis et al., 1999; Allen et al., 2003). Acute nicotine administration has been shown to rapidly and transiently induce c-fos expression in limbic and cortical brain regions (Kiba and Jayaraman, 1994; Salminen et al., 1996, 1999; Seppä et al., 2001; Shram et al., 2007), but this effect is attenuated following continuous administration (Salminen et al., 1999). Thus, it was predicted that prior chronic nicotine exposure would increase expression of c-fos in brain regions also associated with cannabinoid signalling and thus potentiate or attenuate acute THC-induced changes in locomotor activity, effects that would be potentially mediated by age and sex in Sprague-Dawley rats.

    • Immediate early gene expression reveals interactions between social and nicotine rewards on brain activity in adolescent male rats

      2016, Behavioural Brain Research
      Citation Excerpt :

      Contrary to our predictions, we observed less Fos and Zif268 expression in the Cg1, Cg2, dlCPu, and NAcC in nicotine-conditioned rats relative to saline-conditioned rats in Experiment 2. These results are inconsistent with previous findings that have shown acute nicotine administration increases Fos protein and mRNA expression in the cingulate cortex [53–55,89], dorsal striatum [53–55], and ventral striatum, particularly NAcC [53–55,71,89,90]. However, one study reported decreased c-fos and zif268 expression in the frontal cortex, basolateral amygdala and the hippocampus of the mouse brain in response to a high dose of nicotine (1.0 mg/kg, i.p.) [91].

    • Intravenous CDP-choline activates neurons in supraoptic and paraventricular nuclei and induces hormone secretion

      2012, Brain Research Bulletin
      Citation 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.

    View all citing articles on Scopus
    View full text