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Genetic deletion of muscarinic M4 receptors is anxiolytic in the shock-probe burying model
European Journal of Pharmacology 531 (2006) 183 – 186 www.elsevier.com/locate/ejphar
Short communication
Genetic deletion of muscarinic M4 receptors is anxiolytic in the shock-probe burying model Aldemar Degroot, George G. Nomikos ⁎ Eli Lilly and Company, Lilly Corporate Center, Neuroscience Discovery Research, Indianapolis, IN, 46285-0510, USA Received 22 August 2005; received in revised form 29 November 2005; accepted 19 December 2005
Abstract We used muscarinic M2 and M4 receptor knockout (KO) mice to further explore the role of the cholinergic system in anxiety. Using the shockprobe burying model we were able to both assess anxiety and cognition. In this paradigm, an anxiolytic response is reflected by decreased burying behavior. In addition, retention latency depicts long-term memory performance. Whereas muscarinic M2 receptor KO mice did not differ behaviorally from wild-type mice, muscarinic M4 receptor KO mice showed increased anxiolysis, but normal long-term memory compared to wild-type mice. Therefore, muscarinic M4 receptors are of particular significance in anxiety modulation that seems dissociated from changes in long-term memory. © 2005 Elsevier B.V. All rights reserved. Keywords: Muscarinic receptor; Transgenic mouse; Cognition; Animal model; Anxiety
1. Introduction Anxiety-provoking situations increase hippocampal acetylcholine efflux and this neurochemical response can be prevented by the administration of anxiolytic drugs. Specifically, low doses of benzodiazepines and selective serotonin reuptake inhibitors (SSRIs) counteract this anxiety-induced increase in acetylcholine, while not affecting basal levels by themselves (Dazzi et al., 1995; Degroot et al., 2004; Degroot and Nomikos, 2005). Therefore, it is possible that clinically effective anxiolytics exert their clinical actions through a reduction in responsiveness of the cholinergic system and an ensuing decrease in cholinergic neurotransmission (e.g., see Brink et al., 2004; Degroot and Nomikos, 2005). Thus far, it remains unclear which cholinergic receptors may be involved in this effect. In direct relevance, it has been suggested that muscarinic receptors are involved in panic disorder, acute stress disorder, and post-traumatic stress disorder (Battaglia, 2002). Five known types of muscarinic receptors (M1–M5) exist in the brain, which differentially modulate various physiological ⁎ Corresponding author. Tel.: +1 317 433 2541; fax: +1 317 276 5546. E-mail address: [email protected] (G.G. Nomikos). 0014-2999/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2005.12.036
and behavioral functions. Whereas the roles of muscarinic M1 and M2 receptors in anxiety have been pharmacologically examined (McGaughy et al., 2000; Perry et al., 1999; Sarter and Bruno, 1997), the involvement of the other receptor sub-types remains to be elucidated. It appears that muscarinic M1, but not M2, receptors, are involved in the regulation of anxiety. For instance, systemic injections or intracranial infusions of the muscarinic M1 receptor antagonist pirenzepine reduce anxiety in the shock-probe burying test and elevated plus-maze, respectively (Degroot and Nomikos, 2005; Wall et al., 2001). On the other hand, gallamine, a muscarinic M2 receptor antagonist, fails to affect activity in the open field test of anxiety (Sienkiewicz-Jarosz et al., 2000). One issue regarding the pharmacological blockade of muscarinic receptors is that muscarinic ligands lack specificity affecting more than one receptor sub-type. This makes the pharmacological examination of muscarinic receptor sub-types in anxiety challenging. The development of muscarinic receptor knockout (KO) mice offers a viable alternative in examining the role of specific muscarinic receptors in the control of anxiety responses. Admittedly one potential drawback of using KO animals is that the deletion of one gene can result in other compensatory responses (Routtenberg, 1995).
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Since the involvement of the cholinergic system in anxiety modulation can have potentially important therapeutic implications, we wanted to further examine how specific muscarinic receptors mediate this effect. We measured anxiety levels in muscarinic M2 and M4 KO mice. We used muscarinic M2 and M4 KO mice since the muscarinic heteroreceptors (M1, M3, M5, but also M2 and M4) modulate the release of other neurotransmitters, whereas muscarinic autoreceptors (M2 and M4) more specifically control acetylcholine release that is used as a reliable marker of central cholinergic function. Thus, muscarinic M2 and M4 KO mice have been recently shown to differ in aversive memory retention and brain acetylcholine efflux under basal and acetylcholine-evoking conditions (Tzavara et al., 2003). Since a link between aversive memory and anxiety has been proposed (Degroot and Nomikos, 2005) it seems plausible that the muscarinic M2 and M4 KO mice also differ in anxiety levels. 2. Materials and methods 2.1. Subjects We tested 8- to 12-week old male mice with genetic deletions of either the muscarinic M2 or M4 receptors, or wildtype control mice that were strain-, age- and weight-matched (n = 9–10 per group). Mice were originally generated as described in Gomeza et al. (1999) and then backcrossed to C57/BL6 (Taconic) genetic background for at least 10 generations. Mice were group-housed (5 per cage) in polycarbonate cages and the treatment of all animals complied with the NIH guidelines for the use of experimental animals.
time). Animals that failed to make contact with the probe during the first 5 min of the test were not included in the data analysis or the subsequent retention session. The 15-min testing period began immediately following the first contact with the probe. Seven days following the training session, animals were returned to the shock-probe chamber for a 15-min retention session. During the retention session the amount of time it took for the animals to contact the probe was used as a measure of retention latency. All behavioral measures were made by an observer who was blind to the experimental conditions and were analyzed with Student's t-test. 3. Results All animals made contact with the probe during the initial 5 min of the training session. Fig. 1 indicates that muscarinic M4, but not M2, KO mice were less anxious during exposure to the shock-probe test. Specifically, muscarinic M4 KO mice displayed significantly reduced burying behavior compared to wild-type controls [t(1, 16) = 3.03, P b 0.01; Fig. 1A], whereas muscarinic M2 KO mice and wild-type mice had similar overall burying times (P N 0.05; Fig. 1B). Neither the retention latency nor the number of shocks taken by muscarinic M2 and M4 KO mice differed significantly from the values in the wild-type animals (P N 0.05; Fig. 2A–D). Burying levels and cognitive measures were not confounded by activity or reactivity to the probe as reflected by similar still times (Fig. 1) and reactivity scores (Fig. 2). Typically, variations in basal values between experiments are widely observed in the shock-probe model, whereas behavioral
2.2. Behavioral assay Anxiety was assessed in the shock-probe burying test. In this test, mice shocked from a stationary, electrified probe push bedding material from the floor of the experimental chamber toward the shock-probe (i.e., they bury the probe) while avoiding further contacts with the probe (e.g., Treit et al., 1981). In both mice and rats, anxiolytic drugs, such as chlordiazepoxide, decrease burying toward the shock-probe (Degroot and Nomikos, 2004; Tsuda et al., 1988). Conversely, anxiogenic drugs, such as FG7142, increase shock-probe burying (Degroot and Nomikos, 2004; Tsuda et al., 1988). The behavioral testing procedures were the same as those used in previous experiments (e.g., Degroot and Nomikos, 2004). An index of the mouse's reactivity to each shock was scored according to the following four-point scale (Pesold and Treit, 1992): (1) flinch involving only head or forepaw, (2) whole body flinch, with or without slow ambulation away from the probe, (3) whole body flinch, and/or jumping, followed by immediate ambulation away from the probe, and (4) whole body flinch and jump (all four paws in the air), followed by immediate and rapid ambulation (i.e., running) to the opposite end of the chamber. In addition, the total time that the mouse spent immobile (e.g., resting on the floor) during the 15-min testing period was used as an index of general activity (still
Fig. 1. Mean (±SEM) burying and still time in muscarinic M4 and M2 receptor knockout mice (n = 9–10 mice per group). During the training session, muscarinic M4 receptor knockout mice have significantly lower burying scores compared with wild-type mice (A), whereas muscarinic M2 receptor knockout mice and their wild-type controls display similar burying levels (B). Activity scores did not differ between groups. ⁎P b 0.01 compared to wild-type mice.
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Fig. 2. Mean (± SEM) number of shocks, retention latency, and reactivity to the probe in muscarinic M4 and M2 receptor knockout mice (n = 9–10 mice per group). Muscarinic M2 receptor knockout and wild-type mice did not differ in the number of shocks taken during the training session (A) or the latency during the retention session (C). Similarly, muscarinic M4 receptor knockout and wild-type mice took a similar number of shocks (B) and had comparable retention latencies (D). Reactivity scores did not differ between groups.
differences within an experiment are consistent. This likely accounts for the apparent basal differences between the control (wild-type) animals from each experiment, although these differences did not reach statistical significance. In addition, differences in responsivity between the respective wild-type animals have previously been reported (Tzavara et al., 2003). 4. Discussion Recently, the role of the cholinergic system in anxiety modulation has gained considerable attention. In addition to evidence gained from animal models of anxiety, there are also neurochemical indications that clinically effective anxiolytics decrease either basal or stress-evoked hippocampal acetylcholine efflux (e.g., Dazzi et al., 1995; Degroot et al., 2004; Degroot and Nomikos, 2005). Moreover, clinical studies suggest that altered cholinergic neurotransmission may result in the onset or prevention of panic attacks (Battaglia et al., 2001) and generalized anxiety. To date, the importance of the different cholinergic receptors, and specifically the various muscarinic receptor sub-types, in anxiety modulation remains to be elucidated. Here we demonstrate for the first time that muscarinic M4 receptors are involved in the control of anxiety responses. These results were not confounded by decreases in activity, reactivity to the probe, or number of contacts made with the probe. In addition, memory of the probe was likely not a factor, since KO mice had similar retention latencies compared to wild-type mice. In contrast to the results obtained with muscarinic M4 KO mice, muscarinic M2 KO animals did not differ in anxiety modulation compared to wild-type controls. This confirms earlier data obtained with pharmacological
blockade of M2 receptors (Sienkiewicz-Jarosz et al., 2000). The fact that neither muscarinic M4 nor M2 KO mice had altered retention latencies compared to control animals partially conforms to previous data, where genetic deletion of muscarinic M4 receptors did not induce cognitive deficits in a passive avoidance task (Tzavara et al., 2003). We previously demonstrated that muscarinic M4, but not M2 KO, mice had increased basal, but a similar stress-induced, release of acetylcholine in the hippocampus compared to wildtype controls (Tzavara et al., 2003). It has been previously proposed that higher acetylcholine efflux in the hippocampus prior to an anxiety-provoking situation can act as a “coping mechanism” resulting in reduced anxiety levels (Smythe et al., 1998). On the other hand, during exposure to an anxietyprovoking stimulus, a reduction in stress-induced hippocampal acetylcholine efflux may reduce the salience of the aversive stimulus, thereby decreasing anxiety (see, e.g., Degroot and Nomikos, 2005). Therefore, the fact that muscarinic M4 KO mice have an increased basal acetylcholine efflux may account for their reduced anxiety response. Although muscarinic M4 KO mice have similar stress-induced hippocampal acetylcholine levels compared to wild-type mice (Tzavara et al., 2003), a decrease in cholinergic neurotransmission at the postsynaptic muscarinic M4 receptors during an anxiety-provoking situation may further account for the anxiolytic response exhibited by muscarinic M4 KO mice. With the recent implication of the cholinergic system in anxiety regulation, a more detailed examination of the role of this system in anxiety may have important clinical significance. Although today's clinically effective anxiolytics target the cholinergic system indirectly at best, a closer look at drugs that
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directly target the cholinergic system may be warranted. Accordingly, it has been suggested that muscarinic receptors are involved in panic disorder, acute stress disorder, and posttraumatic stress disorder. Thus, the onset of panic attacks can be prevented with anti-muscarinic agents and patients suffering from panic disorder have a higher expression of muscarinic receptors (Battaglia et al., 2001; Van Der Zee and Luiten, 1999). Although it is not currently clear which muscarinic receptors are involved in the above mentioned anxiety disorders, our results indicate that the muscarinic M4 receptors may be of particular clinical significance. While, the muscarinic M1 receptor subtype has also been shown to be involved in the regulation of anxiety based on pharmacological studies, these results may have been confounded by non-specific effects on other receptor sub-types. Also, given that sustained increases of extracellular acetylcholine concentrations, such as those found in muscarinic M4 receptor KO mice (see above), can result in reduced expression of cholinergic receptors (e.g., Li et al., 2003), it is possible that these mice have reduced muscarinic M1 receptor number or sensitivity, which, in turn, may have resulted in the observed anxiolytic effect. However, a reduced expression of muscarinic M1 receptors in M4 receptor KO mice (that in fact has not been reported) seems unlikely given that blockade of M1 receptors results in memory impairments (e.g., Ohnuki and Nomura, 1996), and muscarinic M4 receptor KO and wild-type mice perform equally well on cognitive tasks both in the current and other studies (Tzavara et al., 2003). In this regard, a more detailed examination of the muscarinic heteroreceptors in anxiety modulation is necessary and will be the focus of future studies. Acknowledgements We want to thank J. Hart, L. Rorick and D.L. McKinzie for the mice and the relevant discussions. References Battaglia, M., 2002. Beyond the usual suspects: a cholinergic route for panic attacks. Mol. Psychiatry 7, 239–246. Battaglia, M., Bertella, S., Ogliari, A., Bellodi, L., Smeraldi, E., 2001. Modulation by muscarinic antagonists of the response to carbon dioxide challenge in panic disorder. Arch. Gen. Psychiatry 58, 114–119. Brink, C.B., Viljoen, S.L., de Kock, S.E., Stein, D.J., Harvey, B.H., 2004. Effects of myo-inositol versus fluoxetine and imipramine pretreatments on serotonin 5HT2A and muscarinic acetylcholine receptors in human neuroblastoma cells. Metab. Brain Dis. 19, 51–70. Dazzi, L., Motzo, C., Imperato, A., Serra, M., Gessa, G.L., Biggio, G., 1995. Modulation of basal and stress-induced release of acetylcholine and dopamine in rat brain by abecarnil and imidazenil, two anxioselective
gamma-aminobutyric acidA receptor modulators. J. Pharmacol. Exp. Ther. 273, 241–247. Degroot, A., Nomikos, G.G., 2004. Genetic deletion and pharmacological blockade of CB1 receptors modulates anxiety in the shock-probe burying test. Eur. J. Neurosci. 20, 1059–1064. Degroot, A., Nomikos, G.G., 2005. Fluoxetine disrupts the integration between anxiety and aversive memories. Neuropsychopharmacology 30, 391–400. Degroot, A., Wade, M., Salhoff, C., Davis, R.J., Tzavara, E.T., Nomikos, G.G., 2004. Exposure to an elevated platform increases plasma corticosterone and hippocampal acetylcholine in the rat: reversal by chlordiazepoxide. Eur. J. Pharmacol. 493, 103–109. Gomeza, J., Shannon, H., Kostenis, E., Felder, C., Zhang, L., Brodkin, J., Grinberg, A., Sheng, H., Wess, J., 1999. Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout mice. Proc. Natl. Acad. Sci. U. S. A. 96, 1692–1697. Li, B., Duysen, E.G., Volpicelli-Daley, L.A., Levey, A.I., Lockridge, O., 2003. Regulation of muscarinic acetylcholine receptor function in acetylcholinesterase knockout mice. Pharmacol. Biochem. Behav. 74, 977–986. McGaughy, J., Everitt, B.J., Robbins, T.W., Sarter, M., 2000. The role of cortical cholinergic afferent projections in cognition: impact of new selective immunotoxins. Behav. Brain Res. 115, 251–263. Ohnuki, T., Nomura, Y., 1996. Effects of selective muscarinic antagonists, pirenzepine and AF-DX 116, on passive avoidance tasks in mice. Biol. Pharm. Bull. 19, 814–818. Perry, E., Walker, M., Grace, J., Perry, R., 1999. Acetylcholine in mind: a neurotransmitter correlate of consciousness? Trends Neurosci. 22, 273–280. Pesold, C., Treit, D., 1992. Excitotoxic lesions of the septum produce anxiolytic effects in the elevated plus-maze and shock-probe burying tests. Physiol. Behav. 52, 37–47. Routtenberg, A., 1995. Knockout mouse fault lines. Nature 374, 314–315. Sarter, M., Bruno, J.P., 1997. Cognitive functions of cortical acetylcholine: toward a unifying hypothesis. Brain Res. Brain Res. Rev. 23, 28–46. Sienkiewicz-Jarosz, H., Czlonkowska, A.I., Siemiatkowski, M., Maciejak, P., Szyndler, J., Plaznik, A., 2000. The effects of physostigmine and cholinergic receptor ligands on novelty-induced neophobia. J. Neural. Transm. 107, 1403–1412. Smythe, J.W., Murphy, D., Bhatnagar, S., Timothy, C., Costall, B., 1998. The effects of intrahippocampal scopolamine infusions on anxiety in rats as measured by the black–white box test. Brain Res. Bull. 45, 89–93. Treit, D., Pinel, J.P.J., Fibiger, H.C., 1981. Conditioned defensive burying: a new paradigm for the study of anxiolytic agents. Pharmacol. Biochem. Behav. 15, 619–626. Tsuda, A., Yoshishige, I., Tanaka, M., 1988. The contrasting effects of diazepam and yohimbine on conditioned defensive burying in rats. Psychobiology 16, 213–217. Tzavara, E.T., Bymaster, F.P., Felder, C.C., Wade, M., Gomeza, J., Wess, J., McKinzie, D.L., Nomikos, G.G., 2003. Dysregulated hippocampal acetylcholine neurotransmission and impaired cognition in M2, M4 and M2/M4 muscarinic receptor knockout mice. Mol. Psychiatry 8, 673–679. Van Der Zee, A.E., Luiten, P.G.M., 1999. Muscarinic acetylcholine receptors in the hippocampus, neocortex and amygdala: a review of immunocytochemical localization in relation to learning and memory. Prog. Neurobiol. 58, 409–471. Wall, P.M., Flinn, J., Messier, C., 2001. Infralimbic muscarinic M1 receptors modulate anxiety-like behaviour and spontaneous working memory in mice. Psychopharmacology 155, 58–68.
Short communication
Genetic deletion of muscarinic M4 receptors is anxiolytic in the shock-probe burying model Aldemar Degroot, George G. Nomikos ⁎ Eli Lilly and Company, Lilly Corporate Center, Neuroscience Discovery Research, Indianapolis, IN, 46285-0510, USA Received 22 August 2005; received in revised form 29 November 2005; accepted 19 December 2005
Abstract We used muscarinic M2 and M4 receptor knockout (KO) mice to further explore the role of the cholinergic system in anxiety. Using the shockprobe burying model we were able to both assess anxiety and cognition. In this paradigm, an anxiolytic response is reflected by decreased burying behavior. In addition, retention latency depicts long-term memory performance. Whereas muscarinic M2 receptor KO mice did not differ behaviorally from wild-type mice, muscarinic M4 receptor KO mice showed increased anxiolysis, but normal long-term memory compared to wild-type mice. Therefore, muscarinic M4 receptors are of particular significance in anxiety modulation that seems dissociated from changes in long-term memory. © 2005 Elsevier B.V. All rights reserved. Keywords: Muscarinic receptor; Transgenic mouse; Cognition; Animal model; Anxiety
1. Introduction Anxiety-provoking situations increase hippocampal acetylcholine efflux and this neurochemical response can be prevented by the administration of anxiolytic drugs. Specifically, low doses of benzodiazepines and selective serotonin reuptake inhibitors (SSRIs) counteract this anxiety-induced increase in acetylcholine, while not affecting basal levels by themselves (Dazzi et al., 1995; Degroot et al., 2004; Degroot and Nomikos, 2005). Therefore, it is possible that clinically effective anxiolytics exert their clinical actions through a reduction in responsiveness of the cholinergic system and an ensuing decrease in cholinergic neurotransmission (e.g., see Brink et al., 2004; Degroot and Nomikos, 2005). Thus far, it remains unclear which cholinergic receptors may be involved in this effect. In direct relevance, it has been suggested that muscarinic receptors are involved in panic disorder, acute stress disorder, and post-traumatic stress disorder (Battaglia, 2002). Five known types of muscarinic receptors (M1–M5) exist in the brain, which differentially modulate various physiological ⁎ Corresponding author. Tel.: +1 317 433 2541; fax: +1 317 276 5546. E-mail address: [email protected] (G.G. Nomikos). 0014-2999/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2005.12.036
and behavioral functions. Whereas the roles of muscarinic M1 and M2 receptors in anxiety have been pharmacologically examined (McGaughy et al., 2000; Perry et al., 1999; Sarter and Bruno, 1997), the involvement of the other receptor sub-types remains to be elucidated. It appears that muscarinic M1, but not M2, receptors, are involved in the regulation of anxiety. For instance, systemic injections or intracranial infusions of the muscarinic M1 receptor antagonist pirenzepine reduce anxiety in the shock-probe burying test and elevated plus-maze, respectively (Degroot and Nomikos, 2005; Wall et al., 2001). On the other hand, gallamine, a muscarinic M2 receptor antagonist, fails to affect activity in the open field test of anxiety (Sienkiewicz-Jarosz et al., 2000). One issue regarding the pharmacological blockade of muscarinic receptors is that muscarinic ligands lack specificity affecting more than one receptor sub-type. This makes the pharmacological examination of muscarinic receptor sub-types in anxiety challenging. The development of muscarinic receptor knockout (KO) mice offers a viable alternative in examining the role of specific muscarinic receptors in the control of anxiety responses. Admittedly one potential drawback of using KO animals is that the deletion of one gene can result in other compensatory responses (Routtenberg, 1995).
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Since the involvement of the cholinergic system in anxiety modulation can have potentially important therapeutic implications, we wanted to further examine how specific muscarinic receptors mediate this effect. We measured anxiety levels in muscarinic M2 and M4 KO mice. We used muscarinic M2 and M4 KO mice since the muscarinic heteroreceptors (M1, M3, M5, but also M2 and M4) modulate the release of other neurotransmitters, whereas muscarinic autoreceptors (M2 and M4) more specifically control acetylcholine release that is used as a reliable marker of central cholinergic function. Thus, muscarinic M2 and M4 KO mice have been recently shown to differ in aversive memory retention and brain acetylcholine efflux under basal and acetylcholine-evoking conditions (Tzavara et al., 2003). Since a link between aversive memory and anxiety has been proposed (Degroot and Nomikos, 2005) it seems plausible that the muscarinic M2 and M4 KO mice also differ in anxiety levels. 2. Materials and methods 2.1. Subjects We tested 8- to 12-week old male mice with genetic deletions of either the muscarinic M2 or M4 receptors, or wildtype control mice that were strain-, age- and weight-matched (n = 9–10 per group). Mice were originally generated as described in Gomeza et al. (1999) and then backcrossed to C57/BL6 (Taconic) genetic background for at least 10 generations. Mice were group-housed (5 per cage) in polycarbonate cages and the treatment of all animals complied with the NIH guidelines for the use of experimental animals.
time). Animals that failed to make contact with the probe during the first 5 min of the test were not included in the data analysis or the subsequent retention session. The 15-min testing period began immediately following the first contact with the probe. Seven days following the training session, animals were returned to the shock-probe chamber for a 15-min retention session. During the retention session the amount of time it took for the animals to contact the probe was used as a measure of retention latency. All behavioral measures were made by an observer who was blind to the experimental conditions and were analyzed with Student's t-test. 3. Results All animals made contact with the probe during the initial 5 min of the training session. Fig. 1 indicates that muscarinic M4, but not M2, KO mice were less anxious during exposure to the shock-probe test. Specifically, muscarinic M4 KO mice displayed significantly reduced burying behavior compared to wild-type controls [t(1, 16) = 3.03, P b 0.01; Fig. 1A], whereas muscarinic M2 KO mice and wild-type mice had similar overall burying times (P N 0.05; Fig. 1B). Neither the retention latency nor the number of shocks taken by muscarinic M2 and M4 KO mice differed significantly from the values in the wild-type animals (P N 0.05; Fig. 2A–D). Burying levels and cognitive measures were not confounded by activity or reactivity to the probe as reflected by similar still times (Fig. 1) and reactivity scores (Fig. 2). Typically, variations in basal values between experiments are widely observed in the shock-probe model, whereas behavioral
2.2. Behavioral assay Anxiety was assessed in the shock-probe burying test. In this test, mice shocked from a stationary, electrified probe push bedding material from the floor of the experimental chamber toward the shock-probe (i.e., they bury the probe) while avoiding further contacts with the probe (e.g., Treit et al., 1981). In both mice and rats, anxiolytic drugs, such as chlordiazepoxide, decrease burying toward the shock-probe (Degroot and Nomikos, 2004; Tsuda et al., 1988). Conversely, anxiogenic drugs, such as FG7142, increase shock-probe burying (Degroot and Nomikos, 2004; Tsuda et al., 1988). The behavioral testing procedures were the same as those used in previous experiments (e.g., Degroot and Nomikos, 2004). An index of the mouse's reactivity to each shock was scored according to the following four-point scale (Pesold and Treit, 1992): (1) flinch involving only head or forepaw, (2) whole body flinch, with or without slow ambulation away from the probe, (3) whole body flinch, and/or jumping, followed by immediate ambulation away from the probe, and (4) whole body flinch and jump (all four paws in the air), followed by immediate and rapid ambulation (i.e., running) to the opposite end of the chamber. In addition, the total time that the mouse spent immobile (e.g., resting on the floor) during the 15-min testing period was used as an index of general activity (still
Fig. 1. Mean (±SEM) burying and still time in muscarinic M4 and M2 receptor knockout mice (n = 9–10 mice per group). During the training session, muscarinic M4 receptor knockout mice have significantly lower burying scores compared with wild-type mice (A), whereas muscarinic M2 receptor knockout mice and their wild-type controls display similar burying levels (B). Activity scores did not differ between groups. ⁎P b 0.01 compared to wild-type mice.
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Fig. 2. Mean (± SEM) number of shocks, retention latency, and reactivity to the probe in muscarinic M4 and M2 receptor knockout mice (n = 9–10 mice per group). Muscarinic M2 receptor knockout and wild-type mice did not differ in the number of shocks taken during the training session (A) or the latency during the retention session (C). Similarly, muscarinic M4 receptor knockout and wild-type mice took a similar number of shocks (B) and had comparable retention latencies (D). Reactivity scores did not differ between groups.
differences within an experiment are consistent. This likely accounts for the apparent basal differences between the control (wild-type) animals from each experiment, although these differences did not reach statistical significance. In addition, differences in responsivity between the respective wild-type animals have previously been reported (Tzavara et al., 2003). 4. Discussion Recently, the role of the cholinergic system in anxiety modulation has gained considerable attention. In addition to evidence gained from animal models of anxiety, there are also neurochemical indications that clinically effective anxiolytics decrease either basal or stress-evoked hippocampal acetylcholine efflux (e.g., Dazzi et al., 1995; Degroot et al., 2004; Degroot and Nomikos, 2005). Moreover, clinical studies suggest that altered cholinergic neurotransmission may result in the onset or prevention of panic attacks (Battaglia et al., 2001) and generalized anxiety. To date, the importance of the different cholinergic receptors, and specifically the various muscarinic receptor sub-types, in anxiety modulation remains to be elucidated. Here we demonstrate for the first time that muscarinic M4 receptors are involved in the control of anxiety responses. These results were not confounded by decreases in activity, reactivity to the probe, or number of contacts made with the probe. In addition, memory of the probe was likely not a factor, since KO mice had similar retention latencies compared to wild-type mice. In contrast to the results obtained with muscarinic M4 KO mice, muscarinic M2 KO animals did not differ in anxiety modulation compared to wild-type controls. This confirms earlier data obtained with pharmacological
blockade of M2 receptors (Sienkiewicz-Jarosz et al., 2000). The fact that neither muscarinic M4 nor M2 KO mice had altered retention latencies compared to control animals partially conforms to previous data, where genetic deletion of muscarinic M4 receptors did not induce cognitive deficits in a passive avoidance task (Tzavara et al., 2003). We previously demonstrated that muscarinic M4, but not M2 KO, mice had increased basal, but a similar stress-induced, release of acetylcholine in the hippocampus compared to wildtype controls (Tzavara et al., 2003). It has been previously proposed that higher acetylcholine efflux in the hippocampus prior to an anxiety-provoking situation can act as a “coping mechanism” resulting in reduced anxiety levels (Smythe et al., 1998). On the other hand, during exposure to an anxietyprovoking stimulus, a reduction in stress-induced hippocampal acetylcholine efflux may reduce the salience of the aversive stimulus, thereby decreasing anxiety (see, e.g., Degroot and Nomikos, 2005). Therefore, the fact that muscarinic M4 KO mice have an increased basal acetylcholine efflux may account for their reduced anxiety response. Although muscarinic M4 KO mice have similar stress-induced hippocampal acetylcholine levels compared to wild-type mice (Tzavara et al., 2003), a decrease in cholinergic neurotransmission at the postsynaptic muscarinic M4 receptors during an anxiety-provoking situation may further account for the anxiolytic response exhibited by muscarinic M4 KO mice. With the recent implication of the cholinergic system in anxiety regulation, a more detailed examination of the role of this system in anxiety may have important clinical significance. Although today's clinically effective anxiolytics target the cholinergic system indirectly at best, a closer look at drugs that
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directly target the cholinergic system may be warranted. Accordingly, it has been suggested that muscarinic receptors are involved in panic disorder, acute stress disorder, and posttraumatic stress disorder. Thus, the onset of panic attacks can be prevented with anti-muscarinic agents and patients suffering from panic disorder have a higher expression of muscarinic receptors (Battaglia et al., 2001; Van Der Zee and Luiten, 1999). Although it is not currently clear which muscarinic receptors are involved in the above mentioned anxiety disorders, our results indicate that the muscarinic M4 receptors may be of particular clinical significance. While, the muscarinic M1 receptor subtype has also been shown to be involved in the regulation of anxiety based on pharmacological studies, these results may have been confounded by non-specific effects on other receptor sub-types. Also, given that sustained increases of extracellular acetylcholine concentrations, such as those found in muscarinic M4 receptor KO mice (see above), can result in reduced expression of cholinergic receptors (e.g., Li et al., 2003), it is possible that these mice have reduced muscarinic M1 receptor number or sensitivity, which, in turn, may have resulted in the observed anxiolytic effect. However, a reduced expression of muscarinic M1 receptors in M4 receptor KO mice (that in fact has not been reported) seems unlikely given that blockade of M1 receptors results in memory impairments (e.g., Ohnuki and Nomura, 1996), and muscarinic M4 receptor KO and wild-type mice perform equally well on cognitive tasks both in the current and other studies (Tzavara et al., 2003). In this regard, a more detailed examination of the muscarinic heteroreceptors in anxiety modulation is necessary and will be the focus of future studies. Acknowledgements We want to thank J. Hart, L. Rorick and D.L. McKinzie for the mice and the relevant discussions. References Battaglia, M., 2002. Beyond the usual suspects: a cholinergic route for panic attacks. Mol. Psychiatry 7, 239–246. Battaglia, M., Bertella, S., Ogliari, A., Bellodi, L., Smeraldi, E., 2001. Modulation by muscarinic antagonists of the response to carbon dioxide challenge in panic disorder. Arch. Gen. Psychiatry 58, 114–119. Brink, C.B., Viljoen, S.L., de Kock, S.E., Stein, D.J., Harvey, B.H., 2004. Effects of myo-inositol versus fluoxetine and imipramine pretreatments on serotonin 5HT2A and muscarinic acetylcholine receptors in human neuroblastoma cells. Metab. Brain Dis. 19, 51–70. Dazzi, L., Motzo, C., Imperato, A., Serra, M., Gessa, G.L., Biggio, G., 1995. Modulation of basal and stress-induced release of acetylcholine and dopamine in rat brain by abecarnil and imidazenil, two anxioselective
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