Behavioural Brain Research 270 (2014) 256–260
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short Communication
Insular muscarinic signaling regulates anxiety-like behaviors in rats on the elevated plus-maze Hui Li a , Lei Chen a , Peng Li b , Xiaohong Wang b , Haifeng Zhai a,∗ a b
National Institute on Drug Dependence, Peking University, Beijing 100191, China School of Chinese Meteria Medica, Beijing University of Chinese Medicine, Beijing 100102, China
h i g h l i g h t s • Insular microinjection and rat elevated plus-maze are the main methods. • Insular mAChR activation by nonselective and M1 -, M4 -selective agonists down-regulate anxiety-like behaviors. • Insular mAChR inhibition by nonselective and M1 -, M4 -selective antagonists up-regulate anxiety-like behaviors.
a r t i c l e
i n f o
Article history: Received 11 February 2014 Received in revised form 1 April 2014 Accepted 12 May 2014 Available online 17 May 2014 Keywords: Insula Anxiety Elevated plus-maze Muscarinic acetylcholine receptor
a b s t r a c t Anxiety is one of the most prevalent neuropsychiatric disorders, and little is known about its pathogenesis. In order to investigate the neural mechanisms of this mental disorder, we used rat behavior in the elevated plus-maze as an animal model of anxiety and the insular cortex (insula) as a brain target. The microinjection of non-selective and selective M1 and M4 muscarinic acetylcholine receptor (mAChR) agonists or antagonists was used to explore whether the insular muscarinic receptor and its subtypes regulate levels of anxiety. The results showed that both non-selective and selective M1 and M4 mAChR agonists increased the time spent on exploring in the open arms, whereas antagonists decreased exploration. Our results indicate that activation of insular mAChRs could produce anxiolytic effects, whereas inhibition of insular mAChRs could increase anxiety. We concluded that the insular muscarinic system plays a role in the modulation of anxiety, and dysfunction of mAChR signaling may be involved in the mechanism of anxiogenesis. © 2014 Elsevier B.V. All rights reserved.
Anxiety is a prevalent and growing mental health problem in the general public. Despite its prevalence, the pathophysiological mechanisms and etiology remain poorly understood. The insular cortex (insula) links emotions to cognitive processes and behavioral responses. Dysfunction of the insula is linked to the likely emergence of anxiety [1]. Functional neuroimaging technique has revealed that people with increased anxiety have increased regional cerebral blood flow in the insula [2]. In addition, patients with generalized anxiety disorder show a reduced activation in the insula after their symptoms are relieved by pharmacological treatment [3]. Moreover, few animal studies also support a role of the insula in anxiety [4,5].
∗ Corresponding author at: National Institute on Drug Dependence, Peking University, 38#, Xueyuan Road, Haidian District, Beijing 100191, China. Tel.: +86 10 82801343; fax: +86 10 62032624. E-mail addresses: [email protected], [email protected], [email protected] (H. Zhai). http://dx.doi.org/10.1016/j.bbr.2014.05.017 0166-4328/© 2014 Elsevier B.V. All rights reserved.
Researches on the muscarinic acetylcholine receptor (mAChR) antagonist scopolamine indicated that systemic usage increases ratings of anxiety in rats [6] and mice [7]. On the other side, systemic injection of the mAChR agonist pilocarpine induces longlasting anxiogenic responses in rats [8]. Studies with scopolamine and pilocarpine indicate that the functional integrity of mAChR is essential for anxiety regulation. It is known that insular mAChR takes part in conditioned taste aversion, attenuation of neophobia, inhibitory avoidance, and object recognition memory [9]. However, the role of insular mAChR and its subtypes (M1 –M5 ) in anxiety is hardly explored. In one of our previous studies [10], we showed that activation of M1 and deactivation of M4 mAChRs up-regulated morphine conditioned place preference (CPP), and that the deactivation of M1 and activation of M4 mAChRs down-regulated CPP expression. Since both the insula and the muscarinic system are respectively play key roles in the anxiety disorder, we wonder whether insular mAChR participate in the modulation of anxiety with a subtype-specific manner. In this study, we adapted a typical anxiety animal model, the elevated plus-maze (EPM) of rats, and the
H. Li et al. / Behavioural Brain Research 270 (2014) 256–260
technology of microinjections of mAChR antagonists and agonists into the insula. On the arrangement, we firstly used the nonselective mAChR agonist and antagonist to validate the involvement of insular mAChR in anxiety. After it was verified, the roles of M1 and M4 subtypes were investigated. Adult male Sprague-Dawley rats (Vital River Laboratory Animal Technology Co. Ltd., Beijing, China), weighing 260–280 g before surgery, were individually housed after surgery in a temperatureand humidity-controlled room (22 ◦ C and ∼60%, respectively) under a 12-h light/dark cycle (lights on at 8:00 A.M.) with ad libitum access to food and water. All rats were allowed to become accustomed to the housing environment for at least 3 days before the stereotaxic surgery. The study procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and approved by the Ethical Committee of Animal Use and Protection of Peking University Health Science Center (No. LA2012-34). The non-selective mAChR agonist (pilocarpine) and antagonist (scopolamine) were respectively provided by Guangzhou Pharmaceutical Co., Ltd and Shanghai Pharmaceutical Supply Station (China). The selective agonists MCN-A-343 (M1 preferring) and LY-2033298 (M4 preferring) and the selective mAChR antagonists pirenzepine (M1 preferring) and tropicamide (M4 preferring) were purchased from Sigma–Aldrich, USA. The LY-2033298 was dissolved in 100% DMSO, tropicamide in 10% DMSO, and all other drugs were prepared in 0.9% saline. Three experiments were carried out in this study. In experiment 1, pilocarpine (0.4 g/site and 0.5 l/site) and scopolamine (30 g/site and 0.5 l/site) were tested. In experiment 2, MCNA-343 (30 ng/site and 0.5 l/site) pirenzepine (21.2 mg/site and 0.5 l/site) were tested. In experiment 3, LY-2033298 (0.4 g/site and 0.5 l/site) and tropicamide (1 mM and 0.5 l/site) were tested. The doses of agonists and antagonists were adapted from one of our previous studies [10] and references [11]. Because the anxiety level in EPMs is positively correlated with light illumination, alternation of light illumination to optimize reference anxiety level can make behavioral observations more effective [12]. In all above three experiments, EPMs with microinjections of agonists and their vehicles were performed under a high illumination condition (150 lx) for assessing anxiolytic effects and EPMs with microinjections of antagonists and their vehicles were performed under a low illumination condition (1 lx) for assessing anxiogenic effects. All rats were first prepared with implantation of cannula. Rats were intraperitoneally anesthetized using 50 mg/kg pentobarbital sodium and positioned in a stereotaxic device (Narishige Co., Japan). Two stainless steel guide cannulas (length = 8 mm, outer diameter = 0.56 mm) were implanted bilaterally and aimed at the insula (AP: −2.3 mm, ML: ±6.5 mm, DV: −6.5 mm) [4]. The guide cannula was fixed to the skull with three anchoring screws and dental cement and a stainless stylet was introduced into each guide cannula to prevent occlusion. After completing the surgery, antibiotic penicillin (75000 U) was intraperitoneally administered for 3–5 days to prevent possible infections. One week after surgery, rats received bilateral infusions into the insula with needles introduced through the guide cannulas until their tips were 1 mm beyond the cannula end. A volume 0.5 l/site of either the vehicle or mAChR agonists/antagonists was injected over 60 s, using two microsyringes connected to an infusion pump. The injection needles were left in place for an additional 60 s to allow for infusion into the brain tissue before removal. The EPM was used to detect anxiety-like behavior in the rats. The apparatus was made of black Plexiglas, had four 50 cm × 10 cm arms, and was elevated 50 cm above the floor. The two closed arms were enclosed by 40 cm walls; the two open arms were surrounded by a 1-cm-high Plexiglas ledge, and all arms were illuminated equally. A 10 cm × 10 cm platform (the junction area of the four
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Fig. 1. (A) An illustration of the microinjection cannula track into the insula; (B) representative location of microinjection sites in the insula in experiment 1, indicated by solid black circles.
arms) at the center was considered the neutral area. Before each test, rats were placed in the testing room for at least 1 h to habituate to the test environment. When performing the test, the rats were put in the center of the maze facing one open arm and were allowed to explore the plus-maze for 5 min. An arm entry was defined as an animal entering the arm with its front two feet [13]. EPM tests were performed 5 min after the insular microinjection [14]. Locomotor activity was assessed in a clean wooden open field arena (80 cm × 80 cm × 80 cm). The floor was marked with lines forming 16 cm × 16 cm squares. When performing this test, the rats were initially placed in the center of the arena for 5 min, and the crossing of any of the lines with all four paws was recorded as locomotor activity. The locomotor activity tests were performed immediately after the EPM tests and under the same illumination conditions. On completion of the behavioral testing, rats were immediately anesthetized with a lethal dose of pentobarbital sodium and 0.5 l/site of 1% Evans blue was microinjected to mark the drug infusion sites. Then the rats were transcardially perfused with 0.1 M phosphate buffer followed by 4% paraformaldehyde. Each rat brain was removed and immersed in 4% paraformaldehyde overnight at 4 ◦ C before sectioning. Fig. 1 shows photomicrographs of representative infusion placement sites in the insula in experiment 1. Rats receiving drug infusions outside this region were excluded from the analysis. Data are expressed as the mean ± SEM. Means were compared with Student’s t test. The statistical significance threshold adopted was P < 0.05. Experiment 1 was first performed with microinjections of the non-selective mAChR agonist pilocarpine under high light
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Fig. 2. Effects of the non-selective mAChR agonist pilocarpine (Pilo) and antagonist scopolamine (Scop) in the elevated plus-maze test of rats. (A) Time spent in open arms; (B) open arm entries; (C) locomotor activity. Tests with pilocarpine and its vehicle were performed under high light illumination. Tests with scopolamine and its vehicle were performed under low light illumination. Each column represents the mean ± SEM of 10–12 rats. *P < 0.05, **P < 0.01 vs. control group.
Fig. 3. Effects of the M1 mAChR agonist MCN-A-343 (MCN) and antagonist pirenzepine (Pire) in the elevated plus-maze test. (A) Time spent in open arms; (B) open arm entries; (C) locomotor activity. Tests with MCN-A-343 and its vehicle were performed under low light illumination. Tests with pirenzepine and its vehicle were performed under low light illumination. Each column represents the mean ± SEM of 10–12 rats. *P < 0.05, **P < 0.01 vs. control group.
illumination. As shown in Fig. 2, pilocarpine significantly increased not only the time rats spent in the open arms (P < 0.01, Fig. 2A), but also the number of open arm entries (P < 0.01, Fig. 2B). The results from the pilocarpine administration illustrated that activation of mAChR decreases the anxiety-like behavior of rats, which disagrees with its anxiogenic effect of systemic administration [8]. Second, the non-selective mAChR antagonist scopolamine was infused under low light illumination. Rats in the scopolamine group spent significantly less time in the open arm than the rats in the vehicle control group (P < 0.05, Fig. 2A), but the number of open arm entries showed no significant difference (Fig. 2B). The results from the scopolamine administration illustrated that inhibition of mAChR increases the anxiety-like behavior of rats, consistent with its anxiogenic effect of systemic administration [6]. Scopolamine or pilocarpine microinjected into the insula produced no significant influence on locomotor activity (Fig. 2C). Similar with the results in experiment 1, the M1 mAChR agonist MCN-A-343 significantly increased the time rats spent in the open arms, whereas the M1 antagonist pirenzepine significantly decreased the time rats spent in the open arms (Ps < 0.05, Fig. 3A). MCN-A-343 showed a trend towards an increased number of open arm entries, but this was not statistically significant (P = 0.059), in contrast to pirenzepine, which significantly decreased the number of open arm entries (P < 0.01, Fig. 3B). As to the locomotor activity, both MCN-A-343 and pirenzepine had no significant effects (Fig. 3C). The results of this experiment indicated that activation of
the M1 mAChR decreases the anxiety-like behavior of rats, whereas inhibition of the M1 mAChR up-regulates the anxiety-like behavior of rats. Similar to the results of experiments 1 and 2, the M4 mAChR agonist LY-2033298 significantly increased the time rats spent in the open arms whereas the M4 antagonist tropicamide significantly decreased the time rats spent in the open arms (Ps < 0.05, Fig. 4A). The results of this experiment indicate that activation of the M4 mAChR decreases the anxiety-like behavior of rats, whereas inhibition of the M4 mAChR up-regulates the anxiety-like behavior of rats. No statistically significant differences were found in the open arm entries (Fig. 4B) or locomotor activity (Fig. 4C). Above results not only provide pharmacological evidence for the insula as a crucial node for anxiety regulation within a rat animal model, but also demonstrate for that insular M1 and M4 receptors are involved in anxiety regulation. Although lots of clinical observations support that the integrity of the insula essential for anxiety modulation [15], hardly any have attempted to prove this using animal studies [4]. Muscarinic signaling has been reported to play a role in anxiety, until now few studies have been able to explore the insular cholinergic system in anxiety (neophobia) [16,17]. Our study presents the successful confirmation that the overall inactivation of insular mAChR with scopolamine increased levels of anxiety and activation of insular mAChR with pilocarpine attenuate anxiety, implicating the pathological mechanism of anxiety may involve the functional deficiency of insular
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Fig. 4. Effects of the selective M4 agonist LY-2033298 (LY) and antagonist tropicamide (Trop) in the elevated plus-maze test. (A) Time spent in open arms; (B) open arm entries; (C) locomotor activity. Tests with LY-2033298 and its vehicle were performed under low light illumination. Tests with tropicamide and its vehicle were performed under low light illumination. Each column represents the mean ± SEM of 10–12 rats. *P < 0.05, **P < 0.01 vs. control group.
mAChR. The insula is well known for taste and object recognition [9]. When rats were exposed to a new taste stimulus, insular acetylcholine increased; and the increase amplitude was parallel with the degree of novelty [18]. Blocking the cholinergic activity with insular microinjection of scopolamine impairs rats’ conditioned taste aversion [19], attenuation of neophobia [17], inhibitory avoidance [14], and object recognition memory [20], four behavioral models involving a learning process of transforming from novelty to familiarity. These findings suggest that insular acetylcholine and mAChR are responsive for decoding novelty-related learning. EPM is generally classified as a model based on the conflict between the tendency to avoid potential danger and the curiosity to explore the novel environment [21]. From a behavioral view of learning, the anxiogenic effects of scopolamine, pirenzepine, and tropicamide in the present study may be due to inhibition of novelty habituation of rats to the open arms in EMP, whereas the anxiolytic effects of pilocarpine, MCN-A-343, and LY-2033298 may be due to enhancement of novelty habituation. A number of studies have implicated mAChR subtypes in the mediation of anxiety. However, the specific role of insular mAChR subtypes in anxiety is not known before the present study. By microinjection into dorsal hippocampus of rats under the social interaction test, M1 antagonist pirenzepine was shown anxiogenic and M2 antagonist gallamine was not significantly anxiogenic or anxiolytic [22], which was partially supported by that M1 receptor agonist McN-A-343 in the dorsal hippocampus was anxiolytic in the EPM test [23]. In the infralimbic cortex of mice under the EPM test, microinjection of scopolamine and M1 agonist McN-A-343 was anxiogenic, whereas M1 antagonist pirenzepine was anxiolytic
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[24]. It was also demonstrated that mice with M4 receptor knockout, but not M2 receptor knock-out, showed increased anxiolysis in shock-probe burying model [25]. These studies, together with our findings, illustrate diverse roles of mAChR subtypes in anxiety modulation – the same subtype positively regulating anxiety in one brain region may mediates opposite effects in other regions. This can give an explanation for why both mAChR antagonist (scopolamine) and agonist (pilocarpine) are anxiogenic when systemically administrated. Few reports deal with the functional role of mAChR subtypes in the insula. By compassion of inhibitive effects of AF DX-116 (an M2 preferring antagonist) and pirenzepine after microinjected into the insula, Naor and Dudai [19] pointed out that conditioned taste aversion of rats was modulated by both M1 and M2 receptors, but M2 subtype was predominant. This finding was not in agreement with a later study, in which Ramirez-Lugo et al. [26] reported that pirenzepine was effective and AF DX-116 was not effective. We ever studied the effects of M1 –M1 antagonists (pirenzepine, methoctramine, 4-DAMP, and tropicamide), M1 agonist MCN-A-343, and M4 agonist LY-2033298 on the morphine-induced CPP [10]. Insular inhibition by microinjection of methoctramine or 4-DAMP had no effects, indicating M2 and M3 subtypes may be not take part in drug memory modulation in the insula. Pirenzepine and LY-2033298 inhibited CPP expression, whereas MCN-A-343 and tropicamide enhanced CPP expression. These findings demonstrated opposite roles of M1 and M4 subtypes in modulation of drug reward memory. However, in this study, both the M1 and M4 mAChR took part in the modulation of anxiety, but showed no functional difference. A hint from the effects of LY-2033298 on CPP and anxiety is that M4 subtype may differently modulate conditioned and unconditioned behaviors. There are several technological notes on this study. The EPM is the most popular of all currently available animal tests of anxiety. The low entries made into the open arms and the short time spent in the open arms are well established to correlate with anxiety [21]. In this study, these two indices were not well linked, and the anxiolytic or anxiogenic effects of antagonists and agonists were mainly judged with reference to the time spent in the open arms. Moreover, it should be noted that this model is easily affected by numerous external factors, such that levels of anxiety are negatively correlated with light illumination [12]. With the aim to make the outcome of antagonists and agonists more observable, we exchanged the light intensity of the testing room for antagonists and agonists. With regard to the microinjection dosing, only the results from the M4 antagonist are in disagreement with previous publications. According to a previous report, 10 M is an effective dose of M4 antagonists [11]. However, this dose produced no anxiolytic or anxiogenic effects in our pilot experiments. Then, we augmented the dose to 100 M and 1 mM, and found that the dose of 1 mM could produce anxiogenic effect (data of 10 M and 100 M are not shown in this paper). The open field test here is used as a behavioral control of the EPM test. The results clearly demonstrate that the drug doses used here are only effective on anxiety-like behavior, and do not act deleteriously on locomotor activity. Our study made no strict anatomical limitations with regard to insular subregions, although anterior and posterior insula are believed to contribute to different functions [27]. Lastly, mood is often multidimensional. Any animal model of anxiety, such as EPM or light-dark box, cannot reveal all factors related to anxiety. In order to make the conclusion more concrete and comprehensive, our findings are still needed to be testified in other anxiety models than EPM. Conclusively, our results provide direct evidence that the insular muscarinic cholinergic system plays a determinant role in the modulation of anxiety. Inhibition of the mAChR and its M1 and M4 subtypes increases levels of anxiety, whereas activation of the
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mAChR and its M1 and M4 subtypes decreases levels of anxiety. These findings suggest that dysfunction of insular mAChR signaling may serve as a mechanism of anxiety. Acknowledgements Fundings for this study were provided by the National Natural Science Foundation (No. 30873051, 81271473) and Beijing Municipal Natural Science Foundation (No. 7112087) of China. References [1] Nieuwenhuys R. The insular cortex: a review. Prog Brain Res 2012;195:123–63. [2] Nakao T, Sanematsu H, Yoshiura T, Togao O, Murayama K, Tomita M, et al. fMRI of patients with social anxiety disorder during a social situation task. Neurosci Res 2011;69:67–72. [3] Hoehn-Saric R, Schlund MW, Wong SH. Effects of citalopram on worry and brain activation in patients with generalized anxiety disorder. Psychiatry Res 2004;131:11–21. [4] Christianson JP, Jennings JH, Ragole T, Flyer JG, Benison AM, Barth DS, et al. Safety signals mitigate the consequences of uncontrollable stress via a circuit involving the sensory insular cortex and bed nucleus of the stria terminalis. Biol Psychiatry 2011;70:458–64. [5] Cohen H, Kozlovsky N, Matar MA, Kaplan Z, Zohar J. Mapping the brain pathways of traumatic memory: inactivation of protein kinase M zeta in different brain regions disrupts traumatic memory processes and attenuates traumatic stress responses in rats. Eur Neuropsychopharmacol 2010;20:253–71. [6] Smythe JW, Murphy D, Bhatnagar S, Timothy C, Costall B. Muscarinic antagonists are anxiogenic in rats tested in the black-white box. Pharmacol Biochem Behav 1996;54:57–63. [7] Rodgers RJ, Cole JC. Effects of scopolamine and its quaternary analogue in the murine elevated plus-maze test of anxiety. Behav Pharmacol 1995;6:283–9. [8] Duarte FS, Gavioli EC, Duzzioni M, Hoeller AA, Canteras NS, De Lima TC. Shortand long-term anxiogenic effects induced by a single injection of subconvulsant doses of pilocarpine in rats: investigation of the putative role of hippocampal pathways. Psychopharmacology (Berl) 2010;212:653–61. [9] Bermudez-Rattoni F. The forgotten insular cortex: its role on recognition memory formation. Neurobiol Learn Mem 2014;109c:207–16. [10] Wu W, Li H, Liu Y, Huang XJ, Chen L, Zha HF. Involvement of insular muscarinic cholinergic receptors in morphine-induced conditioned place preference in rats. Psychopharmacology (Berl) 2014, http://dx.doi.org/10.1007/ s00213-014-3550-1. [11] Bosch D, Schmid S. Activation of muscarinic cholinergic receptors inhibits giant neurones in the caudal pontine reticular nucleus. Eur J Neurosci 2006;24:1967–75.
[12] Bouwknecht JA, Spiga F, Staub DR, Hale MW, Shekhar A, Lowry CA. Differential effects of exposure to low-light or high-light open-field on anxiety-related behaviors: relationship to c-Fos expression in serotonergic and non-serotonergic neurons in the dorsal raphe nucleus. Brain Res Bull 2007;72:32–43. [13] Vorhees CV, Morford LR, Graham DL, Skelton MR, Williams MT. Effects of periadolescent fluoxetine and paroxetine on elevated plus-maze, acoustic startle, and swimming immobility in rats while on and off-drug. Behav Brain Funct 2011;7:41. [14] Miranda MI, Bermudez-Rattoni F. Cholinergic activity in the insular cortex is necessary for acquisition and consolidation of contextual memory. Neurobiol Learn Mem 2007;87:343–51. [15] Paulus MP, Stein MB. An insular view of anxiety. Biol Psychiatry 2006;60:383–7. [16] Gutierrez R, Tellez LA, Bermudez-Rattoni F. Blockade of cortical muscarinic but not NMDA receptors prevents a novel taste from becoming familiar. Eur J Neurosci 2003;17:1556–62. [17] Gutierrez R, Rodriguez-Ortiz CJ, De La Cruz V, Nunez-Jaramillo L, BermudezRattoni F. Cholinergic dependence of taste memory formation: evidence of two distinct processes. Neurobiol Learn Mem 2003;80:323–31. [18] Miranda MI, Ramirez-Lugo L, Bermudez-Rattoni F. Cortical cholinergic activity is related to the novelty of the stimulus. Brain Res 2000;882:230–5. [19] Naor C, Dudai Y. Transient impairment of cholinergic function in the rat insular cortex disrupts the encoding of taste in conditioned taste aversion. Behav Brain Res 1996;79:61–7. [20] Bermudez-Rattoni F, Okuda S, Roozendaal B, McGaugh JL. Insular cortex is involved in consolidation of object recognition memory. Learn Mem 2005;12:447–9. [21] Ramos A. Animal models of anxiety: do I need multiple tests. Trends Pharmacol Sci 2008;29:493–8. [22] File SE, Gonzalez LE, Andrews N. Endogenous acetylcholine in the dorsal hippocampus reduces anxiety through actions on nicotinic and muscarinic1 receptors. Behav Neurosci 1998;112:352–9. [23] File SE, Kenny PJ, Cheeta S. The role of the dorsal hippocampal serotonergic and cholinergic systems in the modulation of anxiety. Pharmacol Biochem Behav 2000;66:65–72. [24] Wall PM, Flinn J, Messier C. Infralimbic muscarinic M1 receptors modulate anxiety-like behaviour and spontaneous working memory in mice. Psychopharmacology (Berl) 2001;155:58–68. [25] Degroot A, Nomikos GG. Genetic deletion of muscarinic M4 receptors is anxiolytic in the shock-probe burying model. Eur J Pharmacol 2006;531: 183–6. [26] Ramirez-Lugo L, Miranda MI, Escobar ML, Espinosa E, Bermudez-Rattoni F. The role of cortical cholinergic pre- and post-synaptic receptors in taste memory formation. Neurobiol Learn Mem 2003;79:184–93. [27] Rodgers KM, Benison AM, Klein A, Barth DS. Auditory, somatosensory, and multisensory insular cortex in the rat. Cereb Cortex 2008;18: 2941–51.
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short Communication
Insular muscarinic signaling regulates anxiety-like behaviors in rats on the elevated plus-maze Hui Li a , Lei Chen a , Peng Li b , Xiaohong Wang b , Haifeng Zhai a,∗ a b
National Institute on Drug Dependence, Peking University, Beijing 100191, China School of Chinese Meteria Medica, Beijing University of Chinese Medicine, Beijing 100102, China
h i g h l i g h t s • Insular microinjection and rat elevated plus-maze are the main methods. • Insular mAChR activation by nonselective and M1 -, M4 -selective agonists down-regulate anxiety-like behaviors. • Insular mAChR inhibition by nonselective and M1 -, M4 -selective antagonists up-regulate anxiety-like behaviors.
a r t i c l e
i n f o
Article history: Received 11 February 2014 Received in revised form 1 April 2014 Accepted 12 May 2014 Available online 17 May 2014 Keywords: Insula Anxiety Elevated plus-maze Muscarinic acetylcholine receptor
a b s t r a c t Anxiety is one of the most prevalent neuropsychiatric disorders, and little is known about its pathogenesis. In order to investigate the neural mechanisms of this mental disorder, we used rat behavior in the elevated plus-maze as an animal model of anxiety and the insular cortex (insula) as a brain target. The microinjection of non-selective and selective M1 and M4 muscarinic acetylcholine receptor (mAChR) agonists or antagonists was used to explore whether the insular muscarinic receptor and its subtypes regulate levels of anxiety. The results showed that both non-selective and selective M1 and M4 mAChR agonists increased the time spent on exploring in the open arms, whereas antagonists decreased exploration. Our results indicate that activation of insular mAChRs could produce anxiolytic effects, whereas inhibition of insular mAChRs could increase anxiety. We concluded that the insular muscarinic system plays a role in the modulation of anxiety, and dysfunction of mAChR signaling may be involved in the mechanism of anxiogenesis. © 2014 Elsevier B.V. All rights reserved.
Anxiety is a prevalent and growing mental health problem in the general public. Despite its prevalence, the pathophysiological mechanisms and etiology remain poorly understood. The insular cortex (insula) links emotions to cognitive processes and behavioral responses. Dysfunction of the insula is linked to the likely emergence of anxiety [1]. Functional neuroimaging technique has revealed that people with increased anxiety have increased regional cerebral blood flow in the insula [2]. In addition, patients with generalized anxiety disorder show a reduced activation in the insula after their symptoms are relieved by pharmacological treatment [3]. Moreover, few animal studies also support a role of the insula in anxiety [4,5].
∗ Corresponding author at: National Institute on Drug Dependence, Peking University, 38#, Xueyuan Road, Haidian District, Beijing 100191, China. Tel.: +86 10 82801343; fax: +86 10 62032624. E-mail addresses: [email protected], [email protected], [email protected] (H. Zhai). http://dx.doi.org/10.1016/j.bbr.2014.05.017 0166-4328/© 2014 Elsevier B.V. All rights reserved.
Researches on the muscarinic acetylcholine receptor (mAChR) antagonist scopolamine indicated that systemic usage increases ratings of anxiety in rats [6] and mice [7]. On the other side, systemic injection of the mAChR agonist pilocarpine induces longlasting anxiogenic responses in rats [8]. Studies with scopolamine and pilocarpine indicate that the functional integrity of mAChR is essential for anxiety regulation. It is known that insular mAChR takes part in conditioned taste aversion, attenuation of neophobia, inhibitory avoidance, and object recognition memory [9]. However, the role of insular mAChR and its subtypes (M1 –M5 ) in anxiety is hardly explored. In one of our previous studies [10], we showed that activation of M1 and deactivation of M4 mAChRs up-regulated morphine conditioned place preference (CPP), and that the deactivation of M1 and activation of M4 mAChRs down-regulated CPP expression. Since both the insula and the muscarinic system are respectively play key roles in the anxiety disorder, we wonder whether insular mAChR participate in the modulation of anxiety with a subtype-specific manner. In this study, we adapted a typical anxiety animal model, the elevated plus-maze (EPM) of rats, and the
H. Li et al. / Behavioural Brain Research 270 (2014) 256–260
technology of microinjections of mAChR antagonists and agonists into the insula. On the arrangement, we firstly used the nonselective mAChR agonist and antagonist to validate the involvement of insular mAChR in anxiety. After it was verified, the roles of M1 and M4 subtypes were investigated. Adult male Sprague-Dawley rats (Vital River Laboratory Animal Technology Co. Ltd., Beijing, China), weighing 260–280 g before surgery, were individually housed after surgery in a temperatureand humidity-controlled room (22 ◦ C and ∼60%, respectively) under a 12-h light/dark cycle (lights on at 8:00 A.M.) with ad libitum access to food and water. All rats were allowed to become accustomed to the housing environment for at least 3 days before the stereotaxic surgery. The study procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and approved by the Ethical Committee of Animal Use and Protection of Peking University Health Science Center (No. LA2012-34). The non-selective mAChR agonist (pilocarpine) and antagonist (scopolamine) were respectively provided by Guangzhou Pharmaceutical Co., Ltd and Shanghai Pharmaceutical Supply Station (China). The selective agonists MCN-A-343 (M1 preferring) and LY-2033298 (M4 preferring) and the selective mAChR antagonists pirenzepine (M1 preferring) and tropicamide (M4 preferring) were purchased from Sigma–Aldrich, USA. The LY-2033298 was dissolved in 100% DMSO, tropicamide in 10% DMSO, and all other drugs were prepared in 0.9% saline. Three experiments were carried out in this study. In experiment 1, pilocarpine (0.4 g/site and 0.5 l/site) and scopolamine (30 g/site and 0.5 l/site) were tested. In experiment 2, MCNA-343 (30 ng/site and 0.5 l/site) pirenzepine (21.2 mg/site and 0.5 l/site) were tested. In experiment 3, LY-2033298 (0.4 g/site and 0.5 l/site) and tropicamide (1 mM and 0.5 l/site) were tested. The doses of agonists and antagonists were adapted from one of our previous studies [10] and references [11]. Because the anxiety level in EPMs is positively correlated with light illumination, alternation of light illumination to optimize reference anxiety level can make behavioral observations more effective [12]. In all above three experiments, EPMs with microinjections of agonists and their vehicles were performed under a high illumination condition (150 lx) for assessing anxiolytic effects and EPMs with microinjections of antagonists and their vehicles were performed under a low illumination condition (1 lx) for assessing anxiogenic effects. All rats were first prepared with implantation of cannula. Rats were intraperitoneally anesthetized using 50 mg/kg pentobarbital sodium and positioned in a stereotaxic device (Narishige Co., Japan). Two stainless steel guide cannulas (length = 8 mm, outer diameter = 0.56 mm) were implanted bilaterally and aimed at the insula (AP: −2.3 mm, ML: ±6.5 mm, DV: −6.5 mm) [4]. The guide cannula was fixed to the skull with three anchoring screws and dental cement and a stainless stylet was introduced into each guide cannula to prevent occlusion. After completing the surgery, antibiotic penicillin (75000 U) was intraperitoneally administered for 3–5 days to prevent possible infections. One week after surgery, rats received bilateral infusions into the insula with needles introduced through the guide cannulas until their tips were 1 mm beyond the cannula end. A volume 0.5 l/site of either the vehicle or mAChR agonists/antagonists was injected over 60 s, using two microsyringes connected to an infusion pump. The injection needles were left in place for an additional 60 s to allow for infusion into the brain tissue before removal. The EPM was used to detect anxiety-like behavior in the rats. The apparatus was made of black Plexiglas, had four 50 cm × 10 cm arms, and was elevated 50 cm above the floor. The two closed arms were enclosed by 40 cm walls; the two open arms were surrounded by a 1-cm-high Plexiglas ledge, and all arms were illuminated equally. A 10 cm × 10 cm platform (the junction area of the four
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Fig. 1. (A) An illustration of the microinjection cannula track into the insula; (B) representative location of microinjection sites in the insula in experiment 1, indicated by solid black circles.
arms) at the center was considered the neutral area. Before each test, rats were placed in the testing room for at least 1 h to habituate to the test environment. When performing the test, the rats were put in the center of the maze facing one open arm and were allowed to explore the plus-maze for 5 min. An arm entry was defined as an animal entering the arm with its front two feet [13]. EPM tests were performed 5 min after the insular microinjection [14]. Locomotor activity was assessed in a clean wooden open field arena (80 cm × 80 cm × 80 cm). The floor was marked with lines forming 16 cm × 16 cm squares. When performing this test, the rats were initially placed in the center of the arena for 5 min, and the crossing of any of the lines with all four paws was recorded as locomotor activity. The locomotor activity tests were performed immediately after the EPM tests and under the same illumination conditions. On completion of the behavioral testing, rats were immediately anesthetized with a lethal dose of pentobarbital sodium and 0.5 l/site of 1% Evans blue was microinjected to mark the drug infusion sites. Then the rats were transcardially perfused with 0.1 M phosphate buffer followed by 4% paraformaldehyde. Each rat brain was removed and immersed in 4% paraformaldehyde overnight at 4 ◦ C before sectioning. Fig. 1 shows photomicrographs of representative infusion placement sites in the insula in experiment 1. Rats receiving drug infusions outside this region were excluded from the analysis. Data are expressed as the mean ± SEM. Means were compared with Student’s t test. The statistical significance threshold adopted was P < 0.05. Experiment 1 was first performed with microinjections of the non-selective mAChR agonist pilocarpine under high light
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Fig. 2. Effects of the non-selective mAChR agonist pilocarpine (Pilo) and antagonist scopolamine (Scop) in the elevated plus-maze test of rats. (A) Time spent in open arms; (B) open arm entries; (C) locomotor activity. Tests with pilocarpine and its vehicle were performed under high light illumination. Tests with scopolamine and its vehicle were performed under low light illumination. Each column represents the mean ± SEM of 10–12 rats. *P < 0.05, **P < 0.01 vs. control group.
Fig. 3. Effects of the M1 mAChR agonist MCN-A-343 (MCN) and antagonist pirenzepine (Pire) in the elevated plus-maze test. (A) Time spent in open arms; (B) open arm entries; (C) locomotor activity. Tests with MCN-A-343 and its vehicle were performed under low light illumination. Tests with pirenzepine and its vehicle were performed under low light illumination. Each column represents the mean ± SEM of 10–12 rats. *P < 0.05, **P < 0.01 vs. control group.
illumination. As shown in Fig. 2, pilocarpine significantly increased not only the time rats spent in the open arms (P < 0.01, Fig. 2A), but also the number of open arm entries (P < 0.01, Fig. 2B). The results from the pilocarpine administration illustrated that activation of mAChR decreases the anxiety-like behavior of rats, which disagrees with its anxiogenic effect of systemic administration [8]. Second, the non-selective mAChR antagonist scopolamine was infused under low light illumination. Rats in the scopolamine group spent significantly less time in the open arm than the rats in the vehicle control group (P < 0.05, Fig. 2A), but the number of open arm entries showed no significant difference (Fig. 2B). The results from the scopolamine administration illustrated that inhibition of mAChR increases the anxiety-like behavior of rats, consistent with its anxiogenic effect of systemic administration [6]. Scopolamine or pilocarpine microinjected into the insula produced no significant influence on locomotor activity (Fig. 2C). Similar with the results in experiment 1, the M1 mAChR agonist MCN-A-343 significantly increased the time rats spent in the open arms, whereas the M1 antagonist pirenzepine significantly decreased the time rats spent in the open arms (Ps < 0.05, Fig. 3A). MCN-A-343 showed a trend towards an increased number of open arm entries, but this was not statistically significant (P = 0.059), in contrast to pirenzepine, which significantly decreased the number of open arm entries (P < 0.01, Fig. 3B). As to the locomotor activity, both MCN-A-343 and pirenzepine had no significant effects (Fig. 3C). The results of this experiment indicated that activation of
the M1 mAChR decreases the anxiety-like behavior of rats, whereas inhibition of the M1 mAChR up-regulates the anxiety-like behavior of rats. Similar to the results of experiments 1 and 2, the M4 mAChR agonist LY-2033298 significantly increased the time rats spent in the open arms whereas the M4 antagonist tropicamide significantly decreased the time rats spent in the open arms (Ps < 0.05, Fig. 4A). The results of this experiment indicate that activation of the M4 mAChR decreases the anxiety-like behavior of rats, whereas inhibition of the M4 mAChR up-regulates the anxiety-like behavior of rats. No statistically significant differences were found in the open arm entries (Fig. 4B) or locomotor activity (Fig. 4C). Above results not only provide pharmacological evidence for the insula as a crucial node for anxiety regulation within a rat animal model, but also demonstrate for that insular M1 and M4 receptors are involved in anxiety regulation. Although lots of clinical observations support that the integrity of the insula essential for anxiety modulation [15], hardly any have attempted to prove this using animal studies [4]. Muscarinic signaling has been reported to play a role in anxiety, until now few studies have been able to explore the insular cholinergic system in anxiety (neophobia) [16,17]. Our study presents the successful confirmation that the overall inactivation of insular mAChR with scopolamine increased levels of anxiety and activation of insular mAChR with pilocarpine attenuate anxiety, implicating the pathological mechanism of anxiety may involve the functional deficiency of insular
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Fig. 4. Effects of the selective M4 agonist LY-2033298 (LY) and antagonist tropicamide (Trop) in the elevated plus-maze test. (A) Time spent in open arms; (B) open arm entries; (C) locomotor activity. Tests with LY-2033298 and its vehicle were performed under low light illumination. Tests with tropicamide and its vehicle were performed under low light illumination. Each column represents the mean ± SEM of 10–12 rats. *P < 0.05, **P < 0.01 vs. control group.
mAChR. The insula is well known for taste and object recognition [9]. When rats were exposed to a new taste stimulus, insular acetylcholine increased; and the increase amplitude was parallel with the degree of novelty [18]. Blocking the cholinergic activity with insular microinjection of scopolamine impairs rats’ conditioned taste aversion [19], attenuation of neophobia [17], inhibitory avoidance [14], and object recognition memory [20], four behavioral models involving a learning process of transforming from novelty to familiarity. These findings suggest that insular acetylcholine and mAChR are responsive for decoding novelty-related learning. EPM is generally classified as a model based on the conflict between the tendency to avoid potential danger and the curiosity to explore the novel environment [21]. From a behavioral view of learning, the anxiogenic effects of scopolamine, pirenzepine, and tropicamide in the present study may be due to inhibition of novelty habituation of rats to the open arms in EMP, whereas the anxiolytic effects of pilocarpine, MCN-A-343, and LY-2033298 may be due to enhancement of novelty habituation. A number of studies have implicated mAChR subtypes in the mediation of anxiety. However, the specific role of insular mAChR subtypes in anxiety is not known before the present study. By microinjection into dorsal hippocampus of rats under the social interaction test, M1 antagonist pirenzepine was shown anxiogenic and M2 antagonist gallamine was not significantly anxiogenic or anxiolytic [22], which was partially supported by that M1 receptor agonist McN-A-343 in the dorsal hippocampus was anxiolytic in the EPM test [23]. In the infralimbic cortex of mice under the EPM test, microinjection of scopolamine and M1 agonist McN-A-343 was anxiogenic, whereas M1 antagonist pirenzepine was anxiolytic
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[24]. It was also demonstrated that mice with M4 receptor knockout, but not M2 receptor knock-out, showed increased anxiolysis in shock-probe burying model [25]. These studies, together with our findings, illustrate diverse roles of mAChR subtypes in anxiety modulation – the same subtype positively regulating anxiety in one brain region may mediates opposite effects in other regions. This can give an explanation for why both mAChR antagonist (scopolamine) and agonist (pilocarpine) are anxiogenic when systemically administrated. Few reports deal with the functional role of mAChR subtypes in the insula. By compassion of inhibitive effects of AF DX-116 (an M2 preferring antagonist) and pirenzepine after microinjected into the insula, Naor and Dudai [19] pointed out that conditioned taste aversion of rats was modulated by both M1 and M2 receptors, but M2 subtype was predominant. This finding was not in agreement with a later study, in which Ramirez-Lugo et al. [26] reported that pirenzepine was effective and AF DX-116 was not effective. We ever studied the effects of M1 –M1 antagonists (pirenzepine, methoctramine, 4-DAMP, and tropicamide), M1 agonist MCN-A-343, and M4 agonist LY-2033298 on the morphine-induced CPP [10]. Insular inhibition by microinjection of methoctramine or 4-DAMP had no effects, indicating M2 and M3 subtypes may be not take part in drug memory modulation in the insula. Pirenzepine and LY-2033298 inhibited CPP expression, whereas MCN-A-343 and tropicamide enhanced CPP expression. These findings demonstrated opposite roles of M1 and M4 subtypes in modulation of drug reward memory. However, in this study, both the M1 and M4 mAChR took part in the modulation of anxiety, but showed no functional difference. A hint from the effects of LY-2033298 on CPP and anxiety is that M4 subtype may differently modulate conditioned and unconditioned behaviors. There are several technological notes on this study. The EPM is the most popular of all currently available animal tests of anxiety. The low entries made into the open arms and the short time spent in the open arms are well established to correlate with anxiety [21]. In this study, these two indices were not well linked, and the anxiolytic or anxiogenic effects of antagonists and agonists were mainly judged with reference to the time spent in the open arms. Moreover, it should be noted that this model is easily affected by numerous external factors, such that levels of anxiety are negatively correlated with light illumination [12]. With the aim to make the outcome of antagonists and agonists more observable, we exchanged the light intensity of the testing room for antagonists and agonists. With regard to the microinjection dosing, only the results from the M4 antagonist are in disagreement with previous publications. According to a previous report, 10 M is an effective dose of M4 antagonists [11]. However, this dose produced no anxiolytic or anxiogenic effects in our pilot experiments. Then, we augmented the dose to 100 M and 1 mM, and found that the dose of 1 mM could produce anxiogenic effect (data of 10 M and 100 M are not shown in this paper). The open field test here is used as a behavioral control of the EPM test. The results clearly demonstrate that the drug doses used here are only effective on anxiety-like behavior, and do not act deleteriously on locomotor activity. Our study made no strict anatomical limitations with regard to insular subregions, although anterior and posterior insula are believed to contribute to different functions [27]. Lastly, mood is often multidimensional. Any animal model of anxiety, such as EPM or light-dark box, cannot reveal all factors related to anxiety. In order to make the conclusion more concrete and comprehensive, our findings are still needed to be testified in other anxiety models than EPM. Conclusively, our results provide direct evidence that the insular muscarinic cholinergic system plays a determinant role in the modulation of anxiety. Inhibition of the mAChR and its M1 and M4 subtypes increases levels of anxiety, whereas activation of the
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