European Neuropsychopharmacology (]]]]) ], ]]]–]]]
www.elsevier.com/locate/euroneuro
SHORT COMMUNICATION
Nicotine-induced increase of dopaminergic mesoaccumbal neuron activity is prevented by acute restraint stress. In vivo electrophysiology in rats Francesca Panina,b,n, Alessandra Lintasa,c, Marco Dianaa “G. Minardi” Laboratory of Cognitive Neuroscience, Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy b Brain Repair Centre, University of Cambridge, CB2 0PY Cambridge, United Kingdom c Neuroheuristic Research Group, University of Lausanne, Lausanne, Switzerland a
Received 2 August 2013; received in revised form 10 December 2013; accepted 11 January 2014
KEYWORDS
Abstract
Nicotine; Restraint stress; Electrophysiology; Mesoaccumbal dopaminergic system
Stress is well known to affect responsiveness to drugs of abuse and influencing approaching and drug-taking behaviour in both animals and humans. Consistently, in nicotine addicted subjects both negative events and perceived stress levels are reported to increase drug use and facilitate relapse to smoke even after long periods of abstinence. It has been suggested that stressful stimuli may influence the rewarding properties of abused drugs by acting on the dopaminergic mesolimbic system. In line with this hypothesis, a recent microdialysis study in rats has shown that acute restraint stress exposure prevents the nicotine-induced mesolimbic dopaminergic activation in the nucleus accumbens (NAC) shell via a corticosterone-mediated mechanism. In the present study we sought to evaluate the impact of acute restraint stress on nicotine-induced activation of the mesoaccumbal dopaminergic system by extracellular single unit recordings of antidromicallyidentified NAC shell projecting dopaminergic neurons within the ventral tegmental area (VTA). Nicotine intravenous administration dose-dependently (0.05–0.4 mg/kg) stimulated the spontaneous firing and bursting of mesoaccumbal dopaminergic neurons in unstressed rats, as previously reported. By contrast, nicotine failed to increase mesoaccumbal dopaminergic neuron activity in rats previously exposed to 1-h immobilisation stress. Our observations show that acute restraint stress inhibits the response of the mesoaccumbal dopaminergic system to the stimulating properties of nicotine. These findings corroborate the notion that stress reduces the sensitivity to nicotine and
n
Corresponding author at: John Van Geest Centre for Brain Repair (BRC), University of Cambridge, Forvie Site, Robinson Way, CB2 0PY Cambridge, United Kingdom. Tel.: +44 1223 767 069; fax: +44 1223 331 174. E-mail addresses: [email protected], [email protected] (F. Panin). 0924-977X/$ - see front matter & 2014 Elsevier B.V. and ECNP. All rights reserved. http://dx.doi.org/10.1016/j.euroneuro.2014.01.003 Please cite this article as: Panin, F., et al., Nicotine-induced increase of dopaminergic mesoaccumbal neuron activity is prevented by acute restraint stress. In.... European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.01.003
2
F. Panin et al. suggest that the decreased dopaminergic release in the NAC shell is due to a reduced firing and bursting activity in the VTA. & 2014 Elsevier B.V. and ECNP. All rights reserved.
1.
Introduction
Stress is largely considered as a common denominator among biological, psychological and environmental factors that makes an individual more vulnerable to drug use (Gordon, 2002). Stressful conditions and negative life events facilitate the initiation and maintenance of tobacco smoking (Anda et al., 1999; Morissette et al., 2007) and can alter the behaviour of tobacco addicted individuals by increasing the drug intake and the urge to smoke, as well as precipitating the relapse to smoking behavior (Todd, 2004). Nicotine, the major psychoactive component of tobacco smoke, is considered to be the key compound mediating the primary reinforcement stimulus leading to tobacco addiction (Le Foll and Goldberg, 2006). In animals, footshock stress exposure reinstates nicotine seeking after extinction of the drug-reinforced behavior (Buczek et al., 1999), thereby suggesting that stressors favour nicotine-seeking and taking behaviour. Ample experimental evidences indicate the mesolimbic dopaminergic system and, in particular, the dopaminergic neurons projecting from the ventral tegmental area (VTA) to the medio-ventral subdivision (shell) of the nucleus accumbens (NAC), as a common neural substrate of nicotine and stress. Indeed, similarly to other drugs of abuse, nicotine (Grenhoff et al., 1986; Mereu et al., 1987), as well as cigarette smoke inhalation (Fa et al., 2000), stimulates both the firing and bursting activity of dopaminergic neurons in the VTA, thereby increasing in vivo dopaminergic outflow in the NAC shell (Di Chiara and Imperato, 1988). These stimulating effects are thought to relate to its rewarding and reinforcing effects (Balfour et al., 2000, Corrigall et al., 1992). Moreover, it has been demonstrated that acute stress exposure increases the firing and bursting activity of dopaminergic neurons in the VTA (Shalev et al., 2003), thus suggesting that the dopaminergic mesolimbic pathway could play a key role in the interaction between stress and nicotine. However, a recent in vivo microdialysis study has shown that acute restraint stress exposure prevents the nicotine-induced dopaminergic release in the NAC shell of rats, thereby suggesting that stress may hamper nicotine-induced stimulation of dopaminergic activity (Enrico et al., 2013). On this basis, we investigated the effect of acute restraint stress on the nicotine-induced increase of the dopaminergic mesoaccumbal pathway activity by electrophysiological single cell extracellular recordings from antidromically-identified NAC shell projecting DA neurons within the VTA to evaluate if dopaminergic neurons-spiking activity plays a role in this phenomenon.
2. 2.1.
Experimental procedures Animals
The study was carried out in accordance with current Italian legislation (D.L. 116, 1992) which allows experimentation
on laboratory animals only after submission and approval of a research project to the Ministry of Health (Rome, Italy), and in accordance with European Council directives on the matter (n. 2007/526/CE) and the “Guide for the care and use of laboratory animals” as approved by the Society for Neuroscience (National Research Council, 1996). All efforts were made to minimize animal pain and discomfort and to reduce the number of experimental subjects. All experiments were performed on male albino Wistar rats (Rattus norvegicus) weighting 290–310 g (Harlan Italy). Animals were housed in groups (3–4 per cage) and maintained under controlled environmental conditions (temperature 2272 1C; humidity 60–65%; 12-h light–dark cycle with light from 8:00 to 20:00 h), on a standard laboratory diet and tap water ad libitum. Experiments were performed during the light hours of the cycle, from 9:00 to 18:00 h.
2.2.
Chemicals and drug treatment
All chemicals were of analytical grade obtained from SigmaAldrich, Italy. Nicotine (bitartrate salt) was diluted in unbuffered sterile, apyrogenic sodium chloride 0.9% to a final volume of 0.1 ml and administered intravenously (i.v.) via a silicone catheter (0.3 mm ID, 0.64 mm OD) inserted in the femoral vein. Pharmacological treatments were performed once obtained a stable baseline for antidromically-identified mesoaccumbal VTA dopaminergic neuron firing. Nicotine was administered at exponentially increasing cumulative doses (0.05 to 0.4 mg/kg). Nicotine was used at doses known to elicit significant changes of dopaminergic neuron firing pattern (Mereu et al., 1987) in the VTA. All drug doses were calculated as the free base.
2.3.
Experimental layout and electrophysiology
Rats were randomly assigned to two experimental groups: stress and control. In the stress group, subjects were exposed to immobilization restraint stress for 1 h prior to the electrophysiology experiments by placing the animal in a plexiglass hemicylinder which measured 10 20 cm basis, 4 cm ray and length depending on the animal size. Rats were anaesthetised with urethane (1.3 g/kg, i.p.) and a silicone catheter was inserted into the femoral vein for i.v. drug administration. After exposing the dorsal skull, two holes were drilled for the ipsilateral placement of a recording electrode (glass micropipette TW150F-4, 2–3 mm outer diameter, WPIEurope, Aston Stevenage, UK) in the right VTA (AP= 5.4–5.8; ML=0.4–0.8 mm relative to bregma; DV= 7.0–8.5 mm relative to cortical surface) and a Formvar-coated stimulating stainless steel bipolar electrode (tip diameter, 250 μm) in the NAC shell (AP=1.7; ML=1; DV= 8 mm relative to bregma; see Fig. 1(A) and (B)). Structures were localized according to the stereotaxic atlas of Paxinos and Watson (2005). Single unit activity of dopaminergic neurons located in VTA was recorded extracellularly with glass micropipettes filled with 2% pontamine sky blue (PSB) dissolved in 0.5 M sodium acetate (impedance 4–6 M Ω).
Please cite this article as: Panin, F., et al., Nicotine-induced increase of dopaminergic mesoaccumbal neuron activity is prevented by acute restraint stress. In.... European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.01.003
Nicotine-induced increase of dopaminergic mesoaccumbal neuron activity is prevented by acute restraint stress
3
Fig. 1 (A) Schematic representation of the experimental procedure used for the in vivo antidromic identification and recording of dopaminergic mesoaccumbal neuron activity. The stimulating electrode is placed in the nucleus accumbens (NAC) shell, whereas the recording electrode is placed ipsilaterally in the ventral tegmental area (VTA). (B) Histological verification of stimulation and recording sites. Straight lines in the coronal sections indicate the location of stimulating electrode in the NAC shell. Filled circles indicate the location of recording electrode tips into the VTA. Plates are taken from the Paxinos and Watson (2005) atlas. The number beside each plate corresponds to millimeters from bregma point. (C) Typical feature of a single spike of a dopaminergic neuron (left). Note its long duration (Z2.5 ms, usually) and the triphasic waveform. The spontaneous firing is characterized by two distinct firing patterns: irregularly spaced single spikes (middle) and bursting spikes (right). (D) Representative firing rate histograms of dopaminergic neurons showing that nicotine dose-dependently increases firing rate (top) and that this stimulating effect is prevented by acute restraint stress exposure (bottom). Arrows indicate time of administration. Numbers above arrows indicate cumulative doses expressed in mg/kg.
Please cite this article as: Panin, F., et al., Nicotine-induced increase of dopaminergic mesoaccumbal neuron activity is prevented by acute restraint stress. In.... European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.01.003
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F. Panin et al.
2.4.
Data analysis and statistics
All values are charted as percentages of baseline values and expressed as mean7S.E.M. The average dopaminergic firing rate or burst firing from the baseline were defined as 100%; raw data were then converted into percentages of the baseline. The effect of nicotine on basal firing rate and burst firing for both stress and control group was assessed by a multifactorial analysis of variance (ANOVA) with group as the betweensubjects factor (2 levels: stress and control) and dose as the within-subjects factor (5 levels: from 0 to 0.4 mg/kg). When ANOVA results were significant (po0.05), the post hoc LSD test was performed to allow adequate multiple comparisons between groups. Finally, statistical differences among groups in basal firing rate and burst firing values were assessed by a one-way ANOVA using group as a main factor. All statistics were calculated using Statistica 8.0 for Windows (Statsoft).
3. 3.1.
Results Basal DA neuronal firing and bursting activity
Absolute basal firing rate and burst firing of dopaminergic neurons did not differ across experimental groups (Fig. 2 (A)), as revealed by one-way ANOVA (firing rate [Fgroup (1.30) = 3.28; NS]; %spikes in burst [Fgroup (1.60) = 0.08; NS] and mean spikes/burst [Fgroup (1.20) = 1.17; NS]).
Firing Rate (%)
Firing Rate % Spikes Burst Spikes/Burst
600
CTRL 2.47 ± 0.55 5.33 ± 1.79 2.25 ± 0.20
STRESS 1.36 ± 0.07 6.85 ± 5.39 1.34 ± 0.91
CTRL STRESS
400 200
Spikes in Burst (%)
0
800 600 400 200 0
Spikes/Burst (%)
The extracellular neuronal signal from single neurons was amplified (Neurolog System) and displayed on a digital oscilloscope (Tektronix TDS 3012). The experiments were sampled on line with Spike2 software by a computer connected to CED 1401 interface (Cambridge Electronic Design, Cambridge, UK). VTANAC shell dopaminergic neurons were identified according to the already published criteria (Grace and Bunney, 1983, see Fig. 1(C)) and by antidromic activation. Stimuli consisting of monophasic rectangular pulses (0.1–2.0 mA; 0.1–0.5 ms; 0.8 Hz) generated by a Grass Instrument (Quincy, MA) stimulator (S88) and a stimulus isolation unit (SIU5) and were delivered to the NAC shell through the stimulating electrode. An antidromic response was defined as the ability of evoked spikes to follow stimulation frequencies greater than 250 Hz, displaying constant latency and collision with spontaneously occurring spikes (Lipski, 1981). Bursts were defined as the occurrence of two spikes at inter-spike interval of less than 80 ms and terminated when the inter-spike interval exceeded 160 ms (Grace and Bunney, 1983). The following parameters were determined: firing rate, defined by the number of spikes per second, the burst firing, defined by the total percent of firing occurring in bursts (% spikes in burst) and the mean number of spikes within a burst (mean spikes/burst, Diana et al., 1989). The basal firing rate was recorded for 3 to 5 min prior to nicotine i.v. administration at exponentially increasing cumulative doses (0.05–0.4 mg/kg) at 120 s intervals for each dose (see Fig. 1(D)). At the end of each experiment, the recording site was marked by iontophoretic ejection (20 μA negative current for 15 min) of PSB for histological verification. Only data obtained from animals with correctly implanted electrodes were included in the results (see Fig. 1(B)).
800 600 400 200 0
0 0.05 0.1 0.2 0.4 Cumulative Doses Nicotine (mg/Kg)
Fig. 2 Acute stress exposure prevents the nicotine induced stimulation of DA activity: curves displaying the dose response to nicotine in unstressed (CTRL: filled circles, n =6) and stressed group (STRESS: open circles, n =5) on the firing rate (B), the % spikes in burst (C) and the mean spikes/burst (D). Data are expressed as mean7SEM: + po0.05; + + po0.01; +++ po0.001: effect of NIC; npo0.05, nnpo0.01, nnnpo0.001: effect of stress (two-way ANOVA and LSD test). (A) Basal firing rate, % spikes in burst and means spikes/burst in both the unstressed and stressed group. Statistical analysis reveals no difference after acute stress exposure. Data are expressed as mean7SEM (one-way ANOVA).
3.2. Effect of NIC on the firing pattern of mesoaccumbal VTA DA neurons in control rats and rats exposed to 1-h restraint stress The intravenous administrations of nicotine (0.05 to 0.4 mg/ kg) elicited a dose-dependent increase in the firing rate [Fdose (4.24) = 3.56, po0.05] (Fig. 2(B)). NIC induced a marked increase in % of spikes delivered in burst and the mean spikes/burst of mesoaccumbal VTA DA neurons which did not reach statistical significance: [Fdose (4.66) =1.63, NS; Fdose (4.56) = 1.97, NS], respectively (Fig. 2(C) and (D)). Acute
Please cite this article as: Panin, F., et al., Nicotine-induced increase of dopaminergic mesoaccumbal neuron activity is prevented by acute restraint stress. In.... European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.01.003
Nicotine-induced increase of dopaminergic mesoaccumbal neuron activity is prevented by acute restraint stress restraint stress exposure prevented the stimulating effect of nicotine on the firing rate [Fdose group (4.20) = 2.96; po0.05], the % of spikes in burst [Fdose group (4.15) = 3.63; po0.05], and the mean spikes/bursts [Fdose group (4.87) = 3.07; po0.05]. Post hoc analysis revealed that nicotine elicited a stimulating effect only in the unstressed control group for all the parameters measured (Fig. 2(B)–(D)).
4.
Discussion
Our data show that nicotine dose-dependently increases the spontaneous firing rate and the burst firing of NAC shell projecting VTA DA neurons, as previously described (Grenhoff et al., 1986; Mereu et al., 1987). No differences between stress and control group in the basal firing rate is evident at the time of nicotine administration. The most interesting finding of our work is that the nicotine-induced stimulating effect on mesoaccumbal dopaminergic firing and bursting activity is abolished in animals previously exposed to acute restraint stress. These data reveal a decreased sensitivity to nicotine, after acute stress exposure, of the dopaminergic pathway principally involved in rewarding effects of drugs of abuse (Koob and Bloom, 1988; Wise, 1996). Although the mechanisms by which acute restraint stress could desensitize the mesoaccumbal dopaminergic pathway remains to be elucidated, based on the present results it can be hypothesized that a reduction in nicotine rewarding properties induced by acute stress might underlie the influence of stress in the behaviour of tobacco smoking. To further support this hypothesis, a recent in vivo microdialysis study showed that acute exposure to 1 h of restraint stress prevents the nicotine-induced increase in dopaminergic release in the NAC shell of rats (Enrico et al., 2013). With respect to intracerebral microdialysis, monitoring of dopaminergic neuron firing rate and burst firing provides important complementary information, especially when investigating the impact of psychotropic drugs on dopaminergic neuron function, as it has been demonstrated that burst firing is tightly associated to neurotransmitter release (Gonon, 1988). Based on these data, it can be suggested that the stress related increase in tobacco use (Todd, 2004) might result from an unsatisfied compulsive research of the rewarding nicotine effect in stressed individuals. This possibility is in line with the reward deficiency hypothesis (Blum et al., 1996), which proposes that an impairment of the reward pathway, due to functional or genetic deviations, might underlie drug abuse as a way to seek enhanced stimulation of the reward pathways. It is further consistent with the socalled hypodopaminergic state (Melis et al., 2005) which attributes to a low-functioning dopaminergic system a key role in addiction-related behaviours and ‘boosting’ dopaminergic activity a possible therapeutic potential (Diana, 2011). Although these findings are in line with the reward deficiency hypothesis, it has to be considered that the present results elicit from intravenous administration of nicotine, and they might therefore not be relevant to most conditions in which nicotine is self-administered. To further elucidate the impact of stress on the sensitivity to nicotine
5
of mesoaccumbal dopaminergic neurons, additional experiments might be performed by investigating the effect of stress on the stimulating properties of self-administered nicotine on mesoaccumbal dopaminergic neuron activity, in freely-moving conditions. In conclusion, our data suggest that a reduced sensitivity of the dopaminergic mesoaccumbal system to nicotine in stressed individuals could be a possible neural substrate of the stressinduced increase in tobacco seeking behaviour (Todd, 2004).
Role of funding source This work was supported by the grants from the Assessorato Igiene e Sanità (Regione Autonoma della Sardegna) and PRIN 2004 (Ministero della Salute) to MD. The Assessorato Igiene e Sanità and Ministero della Salute have no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
Conflicts of interest All authors declare that they have no conflicts of interest.
Contributors All authors equally contributed to the design and writing of the protocol. Author Francesca Panin wrote the manuscript. Authors Francesca Panin and Alessandra Lintas managed the literature searches and analysis. All authors contributed and have approved the final manuscript.
Acknowledgements We wish to thank Mr Stefano Aramo for his technical support on this study. We also wish to thank Ms Karin Ibba and Mr Giuseppe Palazzolo for their participation in all the experiments.
References Anda, R.F., Croft, J.B., Felitti, V.J., Nordenberg, D., Giles, W.H., Williamson, D.F., Giovino, G.A., 1999. Adverse childhood experiences and smoking during adolescence and adulthood. JAMA 282, 1652–1658. Balfour, D.J., Wright, A.E., Benwell, M.E., Birrell, C.E., 2000. The putative role of extrasynaptic mesolimbic dopamine in the neurobiology of nicotine dependence. Behav. Brain Res. 113, 73–83. Blum, K., Cull, J.G., Braverman, E.R., Comings, D.E., 1996. Reward deficiency syndrome. Am. Sci. 84, 132–145. Buczek, Y., Le, A.D., Wang, A., Stewart, J., Shaham, Y., 1999. Stress reinstates nicotine seeking but not sucrose solution seeking in rats. Psychopharmacology 144, 183–188. Corrigall, W.A., Franklin, K.B., Coen, K.M., Clarke, P.B., 1992. The mesolimbic dopaminergic system is implicated in the reinforcing effects of nicotine. Psychopharmacology 107, 285–289. Diana, M., 2011. The dopamine hypothesis of drug addiction and its potential therapeutic value. Front. Psychiatry 2, 64. Diana, M., Garcia-Munoz, M., Richards, J., Freed, C.R., 1989. Electrophysiological analysis of dopamine cells from the substantia nigra pars compacta of circling rats. Exp. Brain Res. 74, 625–630.
Please cite this article as: Panin, F., et al., Nicotine-induced increase of dopaminergic mesoaccumbal neuron activity is prevented by acute restraint stress. In.... European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.01.003
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Di Chiara, G., Imperato, A., 1988. Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc. Nat. Acad. Sci. U.S.A. 85, 5274–5278. Enrico, P., Sirca, D., Mereu, M., Peana, A.T., Mercante, B., Diana, M., 2013. Acute restraint stress prevents nicotine-induced mesolimbic dopaminergic activation via a corticosterone-mediated mechanism: a microdialysis study in the rat. Drug Alcohol Depend. 127, 8–14. Fa, M., Carcangiu, G., Passino, N., Ghiglieri, V., Gessa, G.L., Mereu, G., 2000. Cigarette smoke inhalation stimulates dopaminergic neurons in rats. Neuroreport 11, 3637–3639. Gonon, F.G., 1988. Nonlinear relationship between impulse flow and dopamine released by rat midbrain dopaminergic neurons as studied by in vivo electrochemistry. Neuroscience 24, 19–28. Gordon, H.W., 2002. Early environmental stress and biological vulnerability to drug abuse. Psychoneuroendocrinology 27, 115–126. Grace, A.A., Bunney, B.S., 1983. Intracellular and extracellular electrophysiology of nigral dopaminergic neurons-1. Identification and characterization. Neuroscience 10, 301–315. Grenhoff, J., Aston-Jones, G., Svensson, T.H., 1986. Nicotinic effects on the firing pattern of midbrain dopamine neurons. Acta Physiol. Scand. 128, 351–358. Le Foll, B., Goldberg, S.R., 2006. Nicotine as a typical drug of abuse in experimental animals and humans. Psychopharmacology 184, 367–381.
Lipski, J., 1981. Antidromic activation of neurones as an analytic tool in the study of the central nervous system. J. Neurosci. Methods 4, 1–32. Koob, G.F., Bloom, F.E., 1988. Cellular and molecular mechanisms of drug dependence. Science 242, 715–723. Melis, M., Spiga, S., Diana, M., 2005. The dopamine hypothesis of drug addiction: hypodopaminergic state. Int. Rev. Neurobiol. 63, 101–154. Mereu, G., Yoon, K.V., Boi, V., Gessa, G.L., Naes, L., Westfall, T.C., 1987. Preferential stimulation of ventral tegmental area dopaminergic neurons by nicotine. Eur. J. Pharmacol. 141, 395–399. Morissette, S.B., Tull, M.T., Gulliver, S.B., Kamholz, B.W., Zimering, R. T., 2007. Anxiety, anxiety disorders, tobacco use, and nicotine: a critical review of interrelation-ships. Psychol. Bull. 133, 245–272. National Research Council, 1996. Guide for the Care and Use of Laboratory Animals, D.C. National Academies Press, Washington. Paxinos, G., Watson, C., 2005. The Rat Brain in Stereotaxic Coordinates, fifth ed. Elsevier Academic Press. Shalev, U., Marinelli, M., Baumann, M.H., Piazza, P.V., Shaham, Y., 2003. The role of corticosterone in food deprivation-induced reinstatement of cocaine seeking in the rat. Psychopharmacology (Berl) 168, 170–176. Todd, M., 2004. Daily processes in stress and smoking: effect of negative events, nicotine dependence and gender. Psychol. Addict. Behav. 18, 131–139. Wise, R.A., 1996. Neurobiology of addiction. Curr. Opin. Neurobiol. 6, 243–251.
Please cite this article as: Panin, F., et al., Nicotine-induced increase of dopaminergic mesoaccumbal neuron activity is prevented by acute restraint stress. In.... European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.01.003
www.elsevier.com/locate/euroneuro
SHORT COMMUNICATION
Nicotine-induced increase of dopaminergic mesoaccumbal neuron activity is prevented by acute restraint stress. In vivo electrophysiology in rats Francesca Panina,b,n, Alessandra Lintasa,c, Marco Dianaa “G. Minardi” Laboratory of Cognitive Neuroscience, Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy b Brain Repair Centre, University of Cambridge, CB2 0PY Cambridge, United Kingdom c Neuroheuristic Research Group, University of Lausanne, Lausanne, Switzerland a
Received 2 August 2013; received in revised form 10 December 2013; accepted 11 January 2014
KEYWORDS
Abstract
Nicotine; Restraint stress; Electrophysiology; Mesoaccumbal dopaminergic system
Stress is well known to affect responsiveness to drugs of abuse and influencing approaching and drug-taking behaviour in both animals and humans. Consistently, in nicotine addicted subjects both negative events and perceived stress levels are reported to increase drug use and facilitate relapse to smoke even after long periods of abstinence. It has been suggested that stressful stimuli may influence the rewarding properties of abused drugs by acting on the dopaminergic mesolimbic system. In line with this hypothesis, a recent microdialysis study in rats has shown that acute restraint stress exposure prevents the nicotine-induced mesolimbic dopaminergic activation in the nucleus accumbens (NAC) shell via a corticosterone-mediated mechanism. In the present study we sought to evaluate the impact of acute restraint stress on nicotine-induced activation of the mesoaccumbal dopaminergic system by extracellular single unit recordings of antidromicallyidentified NAC shell projecting dopaminergic neurons within the ventral tegmental area (VTA). Nicotine intravenous administration dose-dependently (0.05–0.4 mg/kg) stimulated the spontaneous firing and bursting of mesoaccumbal dopaminergic neurons in unstressed rats, as previously reported. By contrast, nicotine failed to increase mesoaccumbal dopaminergic neuron activity in rats previously exposed to 1-h immobilisation stress. Our observations show that acute restraint stress inhibits the response of the mesoaccumbal dopaminergic system to the stimulating properties of nicotine. These findings corroborate the notion that stress reduces the sensitivity to nicotine and
n
Corresponding author at: John Van Geest Centre for Brain Repair (BRC), University of Cambridge, Forvie Site, Robinson Way, CB2 0PY Cambridge, United Kingdom. Tel.: +44 1223 767 069; fax: +44 1223 331 174. E-mail addresses: [email protected], [email protected] (F. Panin). 0924-977X/$ - see front matter & 2014 Elsevier B.V. and ECNP. All rights reserved. http://dx.doi.org/10.1016/j.euroneuro.2014.01.003 Please cite this article as: Panin, F., et al., Nicotine-induced increase of dopaminergic mesoaccumbal neuron activity is prevented by acute restraint stress. In.... European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.01.003
2
F. Panin et al. suggest that the decreased dopaminergic release in the NAC shell is due to a reduced firing and bursting activity in the VTA. & 2014 Elsevier B.V. and ECNP. All rights reserved.
1.
Introduction
Stress is largely considered as a common denominator among biological, psychological and environmental factors that makes an individual more vulnerable to drug use (Gordon, 2002). Stressful conditions and negative life events facilitate the initiation and maintenance of tobacco smoking (Anda et al., 1999; Morissette et al., 2007) and can alter the behaviour of tobacco addicted individuals by increasing the drug intake and the urge to smoke, as well as precipitating the relapse to smoking behavior (Todd, 2004). Nicotine, the major psychoactive component of tobacco smoke, is considered to be the key compound mediating the primary reinforcement stimulus leading to tobacco addiction (Le Foll and Goldberg, 2006). In animals, footshock stress exposure reinstates nicotine seeking after extinction of the drug-reinforced behavior (Buczek et al., 1999), thereby suggesting that stressors favour nicotine-seeking and taking behaviour. Ample experimental evidences indicate the mesolimbic dopaminergic system and, in particular, the dopaminergic neurons projecting from the ventral tegmental area (VTA) to the medio-ventral subdivision (shell) of the nucleus accumbens (NAC), as a common neural substrate of nicotine and stress. Indeed, similarly to other drugs of abuse, nicotine (Grenhoff et al., 1986; Mereu et al., 1987), as well as cigarette smoke inhalation (Fa et al., 2000), stimulates both the firing and bursting activity of dopaminergic neurons in the VTA, thereby increasing in vivo dopaminergic outflow in the NAC shell (Di Chiara and Imperato, 1988). These stimulating effects are thought to relate to its rewarding and reinforcing effects (Balfour et al., 2000, Corrigall et al., 1992). Moreover, it has been demonstrated that acute stress exposure increases the firing and bursting activity of dopaminergic neurons in the VTA (Shalev et al., 2003), thus suggesting that the dopaminergic mesolimbic pathway could play a key role in the interaction between stress and nicotine. However, a recent in vivo microdialysis study has shown that acute restraint stress exposure prevents the nicotine-induced dopaminergic release in the NAC shell of rats, thereby suggesting that stress may hamper nicotine-induced stimulation of dopaminergic activity (Enrico et al., 2013). On this basis, we investigated the effect of acute restraint stress on the nicotine-induced increase of the dopaminergic mesoaccumbal pathway activity by electrophysiological single cell extracellular recordings from antidromically-identified NAC shell projecting DA neurons within the VTA to evaluate if dopaminergic neurons-spiking activity plays a role in this phenomenon.
2. 2.1.
Experimental procedures Animals
The study was carried out in accordance with current Italian legislation (D.L. 116, 1992) which allows experimentation
on laboratory animals only after submission and approval of a research project to the Ministry of Health (Rome, Italy), and in accordance with European Council directives on the matter (n. 2007/526/CE) and the “Guide for the care and use of laboratory animals” as approved by the Society for Neuroscience (National Research Council, 1996). All efforts were made to minimize animal pain and discomfort and to reduce the number of experimental subjects. All experiments were performed on male albino Wistar rats (Rattus norvegicus) weighting 290–310 g (Harlan Italy). Animals were housed in groups (3–4 per cage) and maintained under controlled environmental conditions (temperature 2272 1C; humidity 60–65%; 12-h light–dark cycle with light from 8:00 to 20:00 h), on a standard laboratory diet and tap water ad libitum. Experiments were performed during the light hours of the cycle, from 9:00 to 18:00 h.
2.2.
Chemicals and drug treatment
All chemicals were of analytical grade obtained from SigmaAldrich, Italy. Nicotine (bitartrate salt) was diluted in unbuffered sterile, apyrogenic sodium chloride 0.9% to a final volume of 0.1 ml and administered intravenously (i.v.) via a silicone catheter (0.3 mm ID, 0.64 mm OD) inserted in the femoral vein. Pharmacological treatments were performed once obtained a stable baseline for antidromically-identified mesoaccumbal VTA dopaminergic neuron firing. Nicotine was administered at exponentially increasing cumulative doses (0.05 to 0.4 mg/kg). Nicotine was used at doses known to elicit significant changes of dopaminergic neuron firing pattern (Mereu et al., 1987) in the VTA. All drug doses were calculated as the free base.
2.3.
Experimental layout and electrophysiology
Rats were randomly assigned to two experimental groups: stress and control. In the stress group, subjects were exposed to immobilization restraint stress for 1 h prior to the electrophysiology experiments by placing the animal in a plexiglass hemicylinder which measured 10 20 cm basis, 4 cm ray and length depending on the animal size. Rats were anaesthetised with urethane (1.3 g/kg, i.p.) and a silicone catheter was inserted into the femoral vein for i.v. drug administration. After exposing the dorsal skull, two holes were drilled for the ipsilateral placement of a recording electrode (glass micropipette TW150F-4, 2–3 mm outer diameter, WPIEurope, Aston Stevenage, UK) in the right VTA (AP= 5.4–5.8; ML=0.4–0.8 mm relative to bregma; DV= 7.0–8.5 mm relative to cortical surface) and a Formvar-coated stimulating stainless steel bipolar electrode (tip diameter, 250 μm) in the NAC shell (AP=1.7; ML=1; DV= 8 mm relative to bregma; see Fig. 1(A) and (B)). Structures were localized according to the stereotaxic atlas of Paxinos and Watson (2005). Single unit activity of dopaminergic neurons located in VTA was recorded extracellularly with glass micropipettes filled with 2% pontamine sky blue (PSB) dissolved in 0.5 M sodium acetate (impedance 4–6 M Ω).
Please cite this article as: Panin, F., et al., Nicotine-induced increase of dopaminergic mesoaccumbal neuron activity is prevented by acute restraint stress. In.... European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.01.003
Nicotine-induced increase of dopaminergic mesoaccumbal neuron activity is prevented by acute restraint stress
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Fig. 1 (A) Schematic representation of the experimental procedure used for the in vivo antidromic identification and recording of dopaminergic mesoaccumbal neuron activity. The stimulating electrode is placed in the nucleus accumbens (NAC) shell, whereas the recording electrode is placed ipsilaterally in the ventral tegmental area (VTA). (B) Histological verification of stimulation and recording sites. Straight lines in the coronal sections indicate the location of stimulating electrode in the NAC shell. Filled circles indicate the location of recording electrode tips into the VTA. Plates are taken from the Paxinos and Watson (2005) atlas. The number beside each plate corresponds to millimeters from bregma point. (C) Typical feature of a single spike of a dopaminergic neuron (left). Note its long duration (Z2.5 ms, usually) and the triphasic waveform. The spontaneous firing is characterized by two distinct firing patterns: irregularly spaced single spikes (middle) and bursting spikes (right). (D) Representative firing rate histograms of dopaminergic neurons showing that nicotine dose-dependently increases firing rate (top) and that this stimulating effect is prevented by acute restraint stress exposure (bottom). Arrows indicate time of administration. Numbers above arrows indicate cumulative doses expressed in mg/kg.
Please cite this article as: Panin, F., et al., Nicotine-induced increase of dopaminergic mesoaccumbal neuron activity is prevented by acute restraint stress. In.... European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.01.003
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2.4.
Data analysis and statistics
All values are charted as percentages of baseline values and expressed as mean7S.E.M. The average dopaminergic firing rate or burst firing from the baseline were defined as 100%; raw data were then converted into percentages of the baseline. The effect of nicotine on basal firing rate and burst firing for both stress and control group was assessed by a multifactorial analysis of variance (ANOVA) with group as the betweensubjects factor (2 levels: stress and control) and dose as the within-subjects factor (5 levels: from 0 to 0.4 mg/kg). When ANOVA results were significant (po0.05), the post hoc LSD test was performed to allow adequate multiple comparisons between groups. Finally, statistical differences among groups in basal firing rate and burst firing values were assessed by a one-way ANOVA using group as a main factor. All statistics were calculated using Statistica 8.0 for Windows (Statsoft).
3. 3.1.
Results Basal DA neuronal firing and bursting activity
Absolute basal firing rate and burst firing of dopaminergic neurons did not differ across experimental groups (Fig. 2 (A)), as revealed by one-way ANOVA (firing rate [Fgroup (1.30) = 3.28; NS]; %spikes in burst [Fgroup (1.60) = 0.08; NS] and mean spikes/burst [Fgroup (1.20) = 1.17; NS]).
Firing Rate (%)
Firing Rate % Spikes Burst Spikes/Burst
600
CTRL 2.47 ± 0.55 5.33 ± 1.79 2.25 ± 0.20
STRESS 1.36 ± 0.07 6.85 ± 5.39 1.34 ± 0.91
CTRL STRESS
400 200
Spikes in Burst (%)
0
800 600 400 200 0
Spikes/Burst (%)
The extracellular neuronal signal from single neurons was amplified (Neurolog System) and displayed on a digital oscilloscope (Tektronix TDS 3012). The experiments were sampled on line with Spike2 software by a computer connected to CED 1401 interface (Cambridge Electronic Design, Cambridge, UK). VTANAC shell dopaminergic neurons were identified according to the already published criteria (Grace and Bunney, 1983, see Fig. 1(C)) and by antidromic activation. Stimuli consisting of monophasic rectangular pulses (0.1–2.0 mA; 0.1–0.5 ms; 0.8 Hz) generated by a Grass Instrument (Quincy, MA) stimulator (S88) and a stimulus isolation unit (SIU5) and were delivered to the NAC shell through the stimulating electrode. An antidromic response was defined as the ability of evoked spikes to follow stimulation frequencies greater than 250 Hz, displaying constant latency and collision with spontaneously occurring spikes (Lipski, 1981). Bursts were defined as the occurrence of two spikes at inter-spike interval of less than 80 ms and terminated when the inter-spike interval exceeded 160 ms (Grace and Bunney, 1983). The following parameters were determined: firing rate, defined by the number of spikes per second, the burst firing, defined by the total percent of firing occurring in bursts (% spikes in burst) and the mean number of spikes within a burst (mean spikes/burst, Diana et al., 1989). The basal firing rate was recorded for 3 to 5 min prior to nicotine i.v. administration at exponentially increasing cumulative doses (0.05–0.4 mg/kg) at 120 s intervals for each dose (see Fig. 1(D)). At the end of each experiment, the recording site was marked by iontophoretic ejection (20 μA negative current for 15 min) of PSB for histological verification. Only data obtained from animals with correctly implanted electrodes were included in the results (see Fig. 1(B)).
800 600 400 200 0
0 0.05 0.1 0.2 0.4 Cumulative Doses Nicotine (mg/Kg)
Fig. 2 Acute stress exposure prevents the nicotine induced stimulation of DA activity: curves displaying the dose response to nicotine in unstressed (CTRL: filled circles, n =6) and stressed group (STRESS: open circles, n =5) on the firing rate (B), the % spikes in burst (C) and the mean spikes/burst (D). Data are expressed as mean7SEM: + po0.05; + + po0.01; +++ po0.001: effect of NIC; npo0.05, nnpo0.01, nnnpo0.001: effect of stress (two-way ANOVA and LSD test). (A) Basal firing rate, % spikes in burst and means spikes/burst in both the unstressed and stressed group. Statistical analysis reveals no difference after acute stress exposure. Data are expressed as mean7SEM (one-way ANOVA).
3.2. Effect of NIC on the firing pattern of mesoaccumbal VTA DA neurons in control rats and rats exposed to 1-h restraint stress The intravenous administrations of nicotine (0.05 to 0.4 mg/ kg) elicited a dose-dependent increase in the firing rate [Fdose (4.24) = 3.56, po0.05] (Fig. 2(B)). NIC induced a marked increase in % of spikes delivered in burst and the mean spikes/burst of mesoaccumbal VTA DA neurons which did not reach statistical significance: [Fdose (4.66) =1.63, NS; Fdose (4.56) = 1.97, NS], respectively (Fig. 2(C) and (D)). Acute
Please cite this article as: Panin, F., et al., Nicotine-induced increase of dopaminergic mesoaccumbal neuron activity is prevented by acute restraint stress. In.... European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.01.003
Nicotine-induced increase of dopaminergic mesoaccumbal neuron activity is prevented by acute restraint stress restraint stress exposure prevented the stimulating effect of nicotine on the firing rate [Fdose group (4.20) = 2.96; po0.05], the % of spikes in burst [Fdose group (4.15) = 3.63; po0.05], and the mean spikes/bursts [Fdose group (4.87) = 3.07; po0.05]. Post hoc analysis revealed that nicotine elicited a stimulating effect only in the unstressed control group for all the parameters measured (Fig. 2(B)–(D)).
4.
Discussion
Our data show that nicotine dose-dependently increases the spontaneous firing rate and the burst firing of NAC shell projecting VTA DA neurons, as previously described (Grenhoff et al., 1986; Mereu et al., 1987). No differences between stress and control group in the basal firing rate is evident at the time of nicotine administration. The most interesting finding of our work is that the nicotine-induced stimulating effect on mesoaccumbal dopaminergic firing and bursting activity is abolished in animals previously exposed to acute restraint stress. These data reveal a decreased sensitivity to nicotine, after acute stress exposure, of the dopaminergic pathway principally involved in rewarding effects of drugs of abuse (Koob and Bloom, 1988; Wise, 1996). Although the mechanisms by which acute restraint stress could desensitize the mesoaccumbal dopaminergic pathway remains to be elucidated, based on the present results it can be hypothesized that a reduction in nicotine rewarding properties induced by acute stress might underlie the influence of stress in the behaviour of tobacco smoking. To further support this hypothesis, a recent in vivo microdialysis study showed that acute exposure to 1 h of restraint stress prevents the nicotine-induced increase in dopaminergic release in the NAC shell of rats (Enrico et al., 2013). With respect to intracerebral microdialysis, monitoring of dopaminergic neuron firing rate and burst firing provides important complementary information, especially when investigating the impact of psychotropic drugs on dopaminergic neuron function, as it has been demonstrated that burst firing is tightly associated to neurotransmitter release (Gonon, 1988). Based on these data, it can be suggested that the stress related increase in tobacco use (Todd, 2004) might result from an unsatisfied compulsive research of the rewarding nicotine effect in stressed individuals. This possibility is in line with the reward deficiency hypothesis (Blum et al., 1996), which proposes that an impairment of the reward pathway, due to functional or genetic deviations, might underlie drug abuse as a way to seek enhanced stimulation of the reward pathways. It is further consistent with the socalled hypodopaminergic state (Melis et al., 2005) which attributes to a low-functioning dopaminergic system a key role in addiction-related behaviours and ‘boosting’ dopaminergic activity a possible therapeutic potential (Diana, 2011). Although these findings are in line with the reward deficiency hypothesis, it has to be considered that the present results elicit from intravenous administration of nicotine, and they might therefore not be relevant to most conditions in which nicotine is self-administered. To further elucidate the impact of stress on the sensitivity to nicotine
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of mesoaccumbal dopaminergic neurons, additional experiments might be performed by investigating the effect of stress on the stimulating properties of self-administered nicotine on mesoaccumbal dopaminergic neuron activity, in freely-moving conditions. In conclusion, our data suggest that a reduced sensitivity of the dopaminergic mesoaccumbal system to nicotine in stressed individuals could be a possible neural substrate of the stressinduced increase in tobacco seeking behaviour (Todd, 2004).
Role of funding source This work was supported by the grants from the Assessorato Igiene e Sanità (Regione Autonoma della Sardegna) and PRIN 2004 (Ministero della Salute) to MD. The Assessorato Igiene e Sanità and Ministero della Salute have no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
Conflicts of interest All authors declare that they have no conflicts of interest.
Contributors All authors equally contributed to the design and writing of the protocol. Author Francesca Panin wrote the manuscript. Authors Francesca Panin and Alessandra Lintas managed the literature searches and analysis. All authors contributed and have approved the final manuscript.
Acknowledgements We wish to thank Mr Stefano Aramo for his technical support on this study. We also wish to thank Ms Karin Ibba and Mr Giuseppe Palazzolo for their participation in all the experiments.
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