Neurochemical Correlates of Nicotine Neurotoxicity on Rat Habenulo-Interpeduncular Cholinergic Neurons

NeuroToxicology 26 (2005) 467–474

Neurochemical Correlates of Nicotine Neurotoxicity on Rat HabenuloInterpeduncular Cholinergic Neurons Elisabetta Ciani 1, Sabina Severi 1, Renata Bartesaghi 1, Antonio Contestabile 2,* 1 2

Department of Human and General Physiology, University of Bologna, Italy Department of Biology, University of Bologna, Via Selmi 3, 40126 Bologna, Italy Received 27 December 2004; accepted 31 March 2005 Available online 4 May 2005

Abstract Chronic administration of high doses of nicotine results in axonal degeneration in the central core of the fasciculus retroflexus, a fiber tract connecting the habenulae (Hb) to the interpeduncular nucleus (IPN). An important part of this connection is cholinergic and neurons of origin are located in the medial Hb. We have undertaken the present investigation in order to ascertain whether the cholinergic Hb–IPN neurons are the actual target of nicotine toxicity and to begin studying molecular correlates of this action. In the present report, we demonstrate that 7-day-long continuous administration of nicotine through osmotic minipumps, results in a significant ( 13%) decrease in the volume of the medial Hb, where cholinergic neurons projecting to the IPN are located, and in a drop of a specific marker for cholinergic neurons, choline acetyltransferase (ChAT), in Hb ( 36%) and IPN ( 28%). At various intervals (2–6 days) during continuous nicotine administration, some apoptotic neurons were visualized in the medial Hb by the TUNEL technique. The chronic nicotine treatment also resulted, after 2 days of continuous administration in significant activation of the transcription factor CREB and the ERK/MAPK survival kinase in the Hb, suggesting that these alterations in expression are in some way related to the neurodegenerative/neuroreparative process. The present observations demonstrate that the cholinergic Hb–IPN neurons are a target for nicotine neurotoxicity and confirm the usefulness of the experimental model used here not only to study the consequences of chronic stimulant abuse, but also to study the neurochemistry of the affected neural systems and the role of signaling factors in neurodegenerative and repair mechanisms. Medical relevance of the data on unique vulnerability of the Hb–IPN connection to nicotine in relation to heavy smoking habits, is briefly discussed.

# 2005 Elsevier Inc. All rights reserved. Keywords: Habenula; Interpeduncular nucleus; Drug of abuse; Choline acetyltransferase; Transcription factor; Survival kinase

INTRODUCTION The habenulo-interpeduncular (Hb–IPN) pathway is at the core of a major route connecting the forebrain to the so-called limbic midbrain centers (Nauta, 1958) and constitutes an important negative feedback control from forebrain cells receiving dopaminergic input, * Corresponding author. Tel.: +39 051 2094134; fax: +39 051 2094286. E-mail address: [email protected] (A. Contestabile).

back onto midbrain dopaminergic neurons (Ellison, 1994, 2002). The anatomical link between these two nuclei, the so-called fasciculus retroflexus of Meynert, as well as the related forebrain pathways have been extensively studied from the anatomical point of view (Herkenham and Nauta, 1979; Marchand et al., 1980; Contestabile and Flumerfelt, 1981; Lenn et al., 1983; Hamill and Jacobowitz, 1984). In parallel with the anatomical organization, the neurochemistry of the connecting pathways has been extensively studied, often giving rise to controversial

0161-813X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2005.04.001

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results (Kataoka et al., 1973; Gottesfeld and Jacobowitz, 1978, 1979; Sastry et al., 1979; Contestabile and Fonnum, 1983; Fonnum and Contestabile, 1984). Recently, it has been shown that prolonged administration of high nicotine dosage results in selective degeneration of axons of the fasciculus retroflexus that, on the basis of their location in the core of the axon bundle, can be putatively identified as originating from neurons of the medial Hb (Carlson et al., 2000, 2001; Ellison, 2002). While actual degeneration of soma of medial Hb neurons has not been, so far, demonstrated, the pattern of axonal degeneration in the fasciculus retroflexus strongly suggests that this model can be used to selectively demonstrate a lesion of the cholinergic Hb–IPN connection. This cholinergic projection has been studied in detail from the anatomical and neurochemical point of view: cholinergic neurons from the medial Hb project to the central core of IPN, which possesses the highest level of cholinergic markers found in the brain (Houser et al., 1983; Wainer et al., 1984; Contestabile et al., 1987). While good evidence supports the existence of a medial Hb–IPN cholinergic pathway (Contestabile and Fonnum, 1983; Villani et al., 1983; Contestabile et al., 1987; Fasolo et al., 1992), this connection may only account for about 50% of the cholinergic activity in both nuclei, the remaining fraction being related to pathways originated in the forebrain septal regions (Gottesfeld and Jacobowitz, 1978, 1979; Contestabile and Fonnum, 1983; Fonnum and Contestabile, 1984). The situation is further complicated by the fact that selective lesions of the cholinergic medial habenular neurons are difficult to obtain, and this raises doubts concerning the actual cholinergic nature of the Hb–IPN connection (Fibiger, 1982; Woolf and Butcher, 1985). We report here that chronic nicotine administration to rats actually results in degeneration of habenular neurons and in overall shrinkage of medial Hb and that this is accompanied by a significant decrease of a specific cholinergic marker in both the Hb and the IPN. It has been recently demonstrated that nicotine induces rapid and transient activation of extracellular signal regulated protein kinase (ERK/MAPK) as well as of cAMP response element binding protein (CREB) in a line of PC12 cells (Nakayama et al., 2001). The same cellular signals were also found to be altered in some brain regions after chronic nicotine administration or withdrawal (Pandey et al., 2001; Brunzell et al., 2003). We, therefore, also investigated the activation of ERK and CREB in the Hb of rat subjected to various schedules and dosages of nicotine administration.

MATERIALS AND METHODS Animals and Surgery Male Wistar rats were purchased from Harlan Italy when approximately 2 months old, kept in our stabulary under veterinary control and used before the end of the third month, when body weight was comprised between 300 and 340 g. Experiments were approved by a local bioethical committee and were done in agreement with the Italian and European Community law on the use of animals for experimental purposes. Schedules of nicotine administration through osmotic minipumps were derived from those previously published and known to result in selective degeneration among fibers of the fasciculus retroflexus (Carlson et al., 2000, 2001). Alzet (Cupertino, CA) osmotic minipumps (2-ML1) with a nominal pumping rate of 9.8 ml/h were filled with a solution of nicotine tartrate in sterile physiological saline and implanted subcutaneously in the back of the animals under light anesthesia. The actual concentration of nicotine tartrate was calculated for each animal on the basis of its weight, in such a way to obtain a steady release of 43.1 mg/kg/day of nicotine tartrate (Sigma, St. Louis, MO), which corresponds to a dose of base equivalents of 15 mg/kg/day. This dosage is known to result in massive degeneration of axons in the central core of the fasciculus retroflexus (Carlson et al., 2000). In order to obtain a maximal degenerative effect, the time course of nicotine delivery was increased to 7 days, compared to the 5 days of administration of the original schedule (Carlson et al., 2000, 2001). During the experiment, implanted animals were hydrated using one or two i.p. injections per day of 5% glucose–saline solution. Control animals were anesthetized and sham-operated. Other animals were treated with minipump delivery for 4 days or with i.p. nicotine tartrate injections at a dose of 11.3 mg/kg (Carlson et al., 2001), in order to study the response of cellular signaling pathways to the drug. This dosage was selected as it was previously shown to result, after 5 days of daily administration, in degeneration of fasciculus retroflexus almost equivalent to the one caused by the minipump administration (Carlson et al., 2001). Single administration or daily administrations in the morning of each day for two or four consecutive days were performed, and the animals were killed 6 h after the single or the last nicotine injection.

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Neurochemistry and Western Blotting For the neurochemical study, at the end of the seventh post-implant day rats were killed by decapitation, the brains were explanted and sliced with a Sorvall tissue chopper. Under the stereomicroscope, the habenulae and the interpeduncular nucleus were microdissected, immediately frozen and kept at 80 8C until assayed. Samples were homogenized in 0.32 M sucrose, added with 0.5% Triton X-100 (final concentration) and the whole homogenate was used to assay protein content (Lowry et al., 1951) and choline acetyltransferase (ChAT) activity. To measure ChAT activity, the method of Fonnum (1975) was used. In brief, aliquots of the homogenates were incubated for 10–20 min at 37 8C in a phosphate buffer (pH 7.4) containing [14C]-labeled acetyl-CoA (NEN-Dupont, specific activity 50 mCi/mmol) at a final concentration of 0.2 mM, choline (8 mM) and eserine (0.1 mM) to inhibit acetylcholinesterase. Reaction was blocked with excess cold phosphate buffer inside scintillation vials and acetylcholine was separated, through gentle shacking, with a ion exchange resin (0.5% sodium tetraphenylborate in acetonitrile), which brought the acetylcholine formed by the enzymatic reaction in solution in the organic phase of the scintillation cocktail used (InstaFluor, Packard). Values of blank samples were subtracted to the radioactivity counts performed by a Beckman scintillation counter and values of enzymatic activity were normalized for the specific activity of the radioactive substrate and protein content of the homogenates. For Western blotting, animals subjected to single or multiple nicotine injections as well as animals implanted with osmotic minipumps for 48 h were used. The Hb were collected by microdissection and lysed by homogenization at 4 8C with a lysis buffer containing 1% deoxycholate, 1 mg/ml aprotinin, 2 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate for 10 min. Lysates were immediately processed by Western blot or kept frozen until assayed. Protein concentration of samples was estimated and equivalent (30 mg) amounts of proteins per sample were subjected to electrophoresis on a 10% sodium dodecyl sulfate-acrylamide gel. The gel was then blotted onto a nitrocellulose membrane and equal loading of protein in each lane was assessed by brief staining of the blot with 0.1% Ponceau S. Blotted membranes were blocked for 1 h in a 4% suspension of dried skimmed milk in PBS and incubated overnight at 4 8C with: (1) a rabbit polyclonal anti-CREB serum; (2) a rabbit serum directed against the phosphorylated

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Ser-133 form of CREB (both from Upstate Biotechnology Inc., Lake Placid, NY; 1:1000 dilution); (3) a polyclonal anti-phospho MAPK serum (New England Biolabs Inc., Beverly, MA; dilution 1:1000); or (4) a polyclonal anti-b-actin serum (Sigma, St. Louis, MO; 1:2000 dilution). Membranes were washed and incubated for 1 h at room temperature with peroxidaseconjugated anti-rabbit immunoglobulin G (IgG) (1:1000 dilution). Specific reactions were revealed with the ECL Western blotting detection reagent (Amersham Corp., Piscataway, NJ). Histology, Morphometry and TUNEL Staining Sham-operated rats or rats carrying the nicotine filled minipump for 7 days, were deeply anesthetized with ether and perfused through the heart with 50 ml buffered saline followed by 350 ml of 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The brain was dissected out and additionally fixed overnight in the same fixative, then washed in buffer and left overnight in the buffer containing 15% sucrose. Fortymicrometer thick sections were obtained at the freezing microtome and every third section was Nissl-stained for subsequent stereological reconstruction of medial Hb. The following stereology system was used: (i) optical microscope (Leitz) equipped with a motorized stage and focus control system; (ii) colour digital videocamera attached to the microscope trinocular tube; and (iii) Image Pro Plus (Media Cybernetics, Silver Spring, MD) with the StagePro module for controlling the motorized stage. Volume measurement was performed by separately tracing the left and right Hb cross-sectional areas. The volume was obtained by multiplying the cross-sectional area of the sections by the distance between sampled sections (120 mm) and summing up the obtained values. Other sham-operated or minipump-carrying rats were perfusion fixed 2, 4 or 6 days after implant and selected sections through the habenular region were treated to reveal apoptotic nuclei with the TUNEL technique (apoptosis kit; Roche, Mannheim, Germany) to detect the presence of dying cells in the medial Hb as a consequence of nicotine administration. Statistics Replicate data were expressed as mean  standard error (S.E.) and were subjected to statistical evaluation of the differences found by using the Student’s unpaired two-tailed test or the Dunnet’s or Bonferroni’s tests after analysis of variance (ANOVA).

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RESULTS Rats chronically exposed to a high nicotine dosage (15 mg of base equivalent/kg/day) through osmotic minipumps for 7 days, were used to determine the decrease of ChAT activity both in the Hb and IPN. In agreement with the presumptive cholinergic nature of the medial Hb–IPN connection running with its axons in the core of fasciculus retroflexus where degenerating axons are found (Contestabile and Fonnum, 1983; Contestabile et al., 1987; Carlson et al., 2000), the treatment resulted in a significant decrease of ChAT activity (Fig. 1) both in the Hb ( 36%) and IPN ( 28%). Morphometric analysis carried out on Nissl-stained sections encompassing the whole length of the habenular nuclei, demonstrated that the treatment resulted in significant decrease ( 13%) of the medial Hb volume, after 7 days of chronic nicotine administration (Fig. 2). To demonstrate that neuronal death in the medial Hb contributed to the shrinkage of the nucleus, we performed TUNEL analysis on sections from brains of rats that had been subjected to the slow nicotine release for 2, 4 or 6 days. While, as expected, no positive cells were ever noticed in the medial Hb of control rats, in many sections from nicotine-treated rats

Fig. 1. Decrease of ChAT activity in the Hb and IPN after 7 days of continuous nicotine release through osmotic minipump. Bars are the mean  S.E. of 10 experiments. *p < 0.05, **p < 0.01 vs. control, Student’s t-test.

Fig. 2. Decrease of medial Hb volume after 7 days of continuous nicotine release. Bars are the mean  S.E. of 10 left and right Hb from five animals per group. *p < 0.05 vs. control, Student’s t-test.

we were able to observe some labeled cells, recognizable on the basis of the dark staining of the condensed nucleus (Fig. 3). Previous studies have suggested that, in other brain regions, nicotine administration could regulate CREB and ERK signaling through phosphorylation (Brunzell et al., 2003). We, therefore, performed experiments able to tell us whether this was also the case for the Hb under different schedules of drug delivery, including nicotine release from osmotic minipumps. As shown by Fig. 4A, one or two daily injections of nicotine were unable to significantly alter, at the 6 h interval, the level of CREB phosphorylation in the Hb. A significant increase of CREB phosphorylation was, instead, demonstrated in the Hb of rats subjected to four daily administrations or implanted with the nicotine-releasing pump from 48 h (Fig. 4A). Concerning ERK, we noticed a similar pattern of kinase activation. Levels of the phosphorylated kinase, indeed, were unchanged in the Hb of rats after one or two daily nicotine administrations, while a clear increase was apparent 6 h after the fourth daily administration of nicotine or after 48 h of continuous release through osmotic minipump (Fig. 4B). Short-term effects of nicotine administrations on CREB and ERK phosphorylation, were stu-

Fig. 3. Presence of some TUNEL-positive cell nuclei (black dots) in the medial Hb, after 2 (b) or 6 (c) days of continuous nicotine release, as compared to a control rat (a).

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Fig. 4. Representative Western blots and densitometry of phosphorylated CREB and ERK in the Hb at different times after single, multiple or continuous nicotine administration. Bars are the mean  S.E. of three experiments. *p < 0.05, **p < 0.01 vs. control, Bonferroni’s test after ANOVA.

died 1 or 3 h after a single injection of nicotine. At both these time intervals, no significant alterations were observed (data not shown).

DISCUSSION The present results characterize at the neurochemical level the degenerative effect of chronic administration of high nicotine dosage, previously detected in the form of massive degeneration of axons localized in the central core of the fasciculus retroflexus (Carlson et al., 2000, 2001), by demonstrating the correlated decrease of ChAT activity in the habenular nucleus and in its target region, the IPN. We further provide evidence for actual death of medial habenular neurons by showing that apoptotic cells are detected at different times during nicotine treatment and that a sizeable decrease of the medial Hb volume takes place after 7 days of treatment. The frequency of TUNEL-positive cells in the medial Hb was small as compared with the measured decrease in the total volume of the nucleus ( 13%). It should be, however, taken into account that apoptotic cells are likely to remain detectable for a relatively short time before being eliminated by microglial phagocytosis. In addition, at least part of nicotine sensitive cells may undergo forms of death different

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from the apoptotic one that is preferentially detected by the TUNEL technique. The parallel decrease of ChAT activity in the Hb and IPN is by itself a reliable quantitative correlate of neuronal loss in the medial Hb, as it is fully compatible with a decreased number of medial habenular neurons derived from the stereologic measurement of the nucleus volume. In a previous study (Fasolo et al., 1992), a decrease in the medial Hb volume of about 30% resulted in decrease of ChAT activity in the Hb and IPN comparable with the one found here. In that study, however, the medial Hb decrease was obtained by interfering with the neurogenetic process of the nucleus and the neuronal loss involved both cholinergic and non-cholinergic neurons. Taken together, and compared with previous data on the localization of degenerating axons in the central core of the fasciculus retroflexus (Carlson et al., 2000, 2001), the present observations constitute an unequivocal demonstration that nicotine administration results in degeneration of medial Hb–IPN cholinergic neurons, well characterized by previous neurochemical studies (Contestabile and Fonnum, 1983; Contestabile et al., 1987; Fasolo et al., 1992). The mechanism(s) of nicotine neurotoxicity towards cholinergic medial Hb neurons is unknown and it is not directly addressed by the present experiments. Nicotine is a well-known activator of nicotinic acetylcholine receptors (nAChRs), in particular of a4b2 and a3b4 subtypes that are enriched in the medial Hb (Carlson et al., 2001; Wada et al., 1989). A wellcharacterized effect of nicotine mediated by nAChRs is the elevation of cellular calcium that may occur both from influx from outside and release from internal stores (Chang and Berg, 2001; Nakayama et al., 2001). Excessive calcium increase may be the basis of the neurotoxic effect of chronic nicotine administration. However, increased levels of free calcium may also result in altered regulation of a number of cellular pathways. It was previously shown in mice that chronic nicotine administration resulted in a decrease of phosphorylated CREB in the nucleus accumbens but in an increase in the prefrontal cortex (Brunzell et al., 2003). In the same region, also ERK phosphorylation was increased by chronic nicotine (Brunzell et al., 2003). In other brain regions, CREB phosphorylation was decreased upon withdrawal after chronic nicotine administration (Pandey et al., 2001). Short-term activation of CREB and ERK upon nicotine stimulation has been demonstrated in several neuronal cells (Chang and Berg, 2001; Nakayama et al., 2001; Hu et al., 2002) and these changes are generally interpreted as mechanisms related to survival and activity-dependent synap-

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tic plasticity (Bito and Takemoto-Kimura, 2003). It may, therefore, seem quite odd that CREB and ERK phosphorylation are increased upon protracted exposure to nicotine and that this appears to be correlated with a neurodegenerative event. Interestingly, a very recent study (Obrietan and Hoyt, 2004) has documented that in transgenic mice that are a model for Huntington’s disease, an increased level of brain CREB phosphorylation and CRE-mediated transcription is present, instead of the expected decrease. In the light of this result, these authors hypothesized that increased CRE-mediated transcription in neuropathological conditions may represent a way to try to counteract neurodegenerative mechanisms (Obrietan and Hoyt, 2004), as activated CREB induces the expression of several survival-promoting and anti-apoptotic genes (Riccio et al., 1999; Ciani et al., 2002; Lonze et al., 2002; Bito and Takemoto-Kimura, 2003). A similar role of ERK-mediated regulation of gene expression has also emerged in several experimental models (Bonni et al., 1999; Impey et al., 1999). Our data do not allow to speculate whether CREB and ERK activation in the Hb after prolonged nicotine exposure are related to a reparative effort of endangered neurons. It is, however, interesting that single acute nicotine administration does not result in increased activation of CREB and ERK in the short run (1–6 h), while multiple, and to an even greater extent, continuous administration is effective. The cells responsible for the increased activity of CREB and ERK in the medial Hb, remain to be determined. The Hb–IPN connection through the fasciculus retroflexus is involved in several important functions, from sleep to analgesia, maternal behavior, feeding, learning, stress response and reward mechanisms and it may also be relevant for some neuropsychiatric disorders (Sutherland and Nakajima, 1981; Sandyk, 1991; Thornton and Davies, 1991; Corodimas et al., 1992; Ma et al., 1992; Valjakka et al., 1998). Noticeably, some of these functions are among those known to be affected by nicotine abuse. The peculiar sensitivity of cholinergic medial Hb neurons to nicotine neurotoxicity, therefore, poses relevant health problems. The dose tested for neurotoxicity in the present study (15 mg/kg/day of nicotine free base) results in levels of plasma nicotine of about 500 ng/ml (Carlson et al., 2001), which is several fold higher than plasma nicotine concentration found in heavy smokers (Wilkins et al., 1982; Benowitz, 1988; Lichtensteiger et al., 1988). However, previous studies have demonstrated substantial degeneration, confined to the rat fasciculus retroflexus fiber tract, for nicotine dosage that resulted

in plasma levels of less than twice those found in heavy smokers (Carlson et al., 2000, 2001). Furthermore, cigarette smoking gives rise to intermittent assumption of the drug and this may result in temporary peaks of nicotine concentration, whose effects would summate over time in the elements of the Hb–IPN circuit (Carlson et al., 2000). While the results obtained in rats should be generalized to smoker with caution (Carlson et al., 2000; Ellison, 2002), the unique vulnerability of the Hb–IPN connection to nicotine suggests that some of the effects of high nicotine assumptions in heavy smokers may receive medical consideration, in relation to adverse effects of smoking that involve functions attributed to this important brain circuit. In conclusion, the present observations confirm the usefulness of the nicotine-induced degeneration of habenulo-interpeduncular neurons not only as a model of ‘‘weak link’’ in brain (Ellison, 2002), but also for studies on neurochemistry of the system as well as on the role of survival and transcriptional factors in its neurodegenerative and repair mechanisms.

ACKNOWLEDGEMENTS The present work was supported by a grant of the University of Bologna in the framework of the special project ‘‘Molecular correlates of pathologies’’. The skilful technical assistance of Miss Monia Bentivogli is gratefully acknowledged.

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