Abstract
Disturbances in central dopaminergic neurotransmission are believed to be centrally involved in the pathogenesis of schizophrenia. Central dopaminergic and cholinergic systems interact and the cholinergic muscarinic agonist xanomeline has shown antipsychotic effects in clinical studies. Preclinical studies indicate that the M4 muscarinic cholinergic receptor subtype (mAChR) modulates the activity of the dopaminergic system and that this specific mAChR subtype is involved in mediating the antipsychotic-like effects of xanomeline. A specific neuronal subpopulation that expresses M4 mAChRs together with D1 dopamine receptors seems to be especially important in modulating dopamine-dependent behaviors. Using mutant mice that lack the M4 mAChR only in D1 dopamine receptor-expressing cells (D1-M4-KO), we investigated the role of this neuronal population in the antipsychotic-like effects of xanomeline in amphetamine-induced hyperactivity and apomorphine-induced climbing. Interestingly, the antipsychotic-like effects of xanomeline in the two models were almost completely abolished in D1-M4-KO mice, suggesting that M4 mAChRs colocalized with D1 dopamine receptors are centrally involved in mediating the antipsychotic-like effects of xanomeline. This is consistent with the hypothesis that activation of the M4 mAChR represents a potential target for the future medical treatment of psychosis.
Introduction
Xanomeline is an M1/M4-preferring muscarinic acetylcholine receptor (mAChR) agonist that, despite its lack of affinity for dopamine receptors, has demonstrated antipsychotic-like effects in rodents (Shannon et al., 2000), antipsychotic effects in schizophrenic patients (Shekhar et al., 2008), and robust effects against psychotic-like behavior in Alzheimer's disease patients (Bodick et al., 1997). Xanomeline has been shown to inhibit apomorphine-induced climbing in mice (Shannon et al., 2000) and amphetamine-induced hyperactivity and apomorphine- and scopolamine-induced disruption of prepulse inhibition in rats and mice (Stanhope et al., 2001; Jones et al., 2005; Thomsen et al., 2010), all models possessing predictive validity for alleviation of psychotic symptoms.
The antipsychotic-like effects of xanomeline have been suggested to be primarily mediated via M4 mAChRs (Bymaster et al., 2002; Woolley et al., 2009; Thomsen et al., 2010). Behavioral and neurochemical analysis of M4 receptor knock-out mice has shown that the M4 mAChR plays an important role in the dopamine homeostasis, especially in striatal areas (Gomeza et al., 1999; Felder et al., 2001; Zhang et al., 2002; Tzavara et al., 2004; Fink-Jensen et al., 2011). The M4 mAChR subtype is the major mAChR expressed in the striatum (Levey, 1993), where the receptor is expressed on cholinergic interneurons, as well as on GABAergic projection neurons, where it is coexpressed with D1 dopamine receptors (Weiner et al., 1990; Bernard et al., 1992). We have recently created a line of mice in which the M4 mAChR is selectively abrogated in D1 dopamine receptor-expressing neurons (D1-M4-KO) and shown that this specific subpopulation of M4 mAChRs is critically involved in the regulation of dopaminergic neurotransmission (Jeon et al., 2010). Here we describe the effects of xanomeline on amphetamine-induced hyperactivity and apomorphine-induced climbing behavior in D1-M4-KO and wild-type mice to investigate the role of this receptor subpopulation in antipsychotic-like effects.
Materials and Methods
Animals.
D1-M4-KO mice were generated as previously described (Jeon et al., 2010). Knock-out mice and their wild-type littermates were bred at the animal facility at the Panum Institute, University of Copenhagen (Copenhagen, Denmark). Mouse genotypes were determined by PCR analysis of mouse ear genomic DNA and genotypes were reconfirmed after the completion of experiments. The mice were housed in standard rodent cages (Macrolon type III), enriched with cardboard housing and nesting material. The animals were kept at room temperature (22–24°C) in a 12 h light/dark cycle (lights on at 6:00 A.M.) with ad libitum access to food and water. All experiments were performed on experimentally naive 10–16 weeks old male mice in the middle of the light cycle (between 9:00 A.M. and 4:00 P.M.). The mice were allowed to acclimatize to the animal facility for at least 7 d before the start of experiments. Animals were taken to the experimental room at least 2 h before initiating the experiment. All procedures were conducted in accordance with guidelines from the Animal Experimentation Inspectorate, Denmark and the European Communities Council Directive of 24 November 1986 (86/609/EEC).
Drugs.
D-amphetamine was obtained from the Copenhagen University Hospital Pharmacy. R-(−)-apomorphine hydrochloride hemihydrate and xanomeline l-tartate hydrate (3-Hexoxy-4-(1-methyl-3,6-dihydro-2H-pyridin-5-yl)-1,2,5-thiadiazole) were purchased from Sigma-Aldrich. All drugs were dissolved in saline and administered by subcutaneous injections (10 ml/kg).
Locomotor activity measurements.
Locomotor activity was assessed in an open-field (40 × 40 × 80 cm) setup, placed in a dimly lit room. A camera located on the ceiling above the open field recorded the experiments. The distance the animal moved was analyzed with the video-tracking program Etho Vison (version 3.1; Noldus). Mice were initially placed in the open field for a 1 h habituation period. Hereafter mice were coadministered with xanomeline (1 and 2 mg/kg), vehicle and amphetamine (2 mg/kg), or vehicle, and locomotor activity was monitored for 2 h.
Apomorphine climbing.
Climbing behavior was measured using the three-point rating scale of Protais et al. (1976). Immediately after an injection of apomorphine (1 or 2 mg/kg), the mice were placed into a cylindrical individual cage (diameter, 14 cm; height, 15 cm; grid-size, 5 × 5 mm) with the top covered by a gray plastic lid and the floor covered by a scant lining of bedding material. An observer who was blinded to drug treatment and genotype measured climbing behavior at 5, 10, 15, 20, 25, and 30 min after apomorphine administration. Climbing behavior was observed for 30 s at the six time points and the score corresponding to the posture the animal adopted the longest was recorded. The following postures were scored: four paws on the floor, 0 points; forefeet holding the grid, 1 point; four paws holding the grid, 2 points. The scores for the six time points were summed (maximum 12 points) and this sum was used in statistical calculations. Xanomeline was administered 25 min before the injection of apomorphine (Shannon et al., 2000).
Data analysis.
Group differences in locomotor and climbing activities were analyzed by two-way ANOVA with genotype and treatment as between-subjects factors, followed by Bonferroni-corrected pairwise comparisons. Within each genotype, treatment groups were analyzed by one-way ANOVA with treatment as the between-subjects factor, followed by Newman–Keuls post hoc test. A p value of <0.05 was considered statistically significant.
Results
Xanomeline does not attenuate amphetamine-induced locomotor activity in D1-M4-KO mice
Overall two-way ANOVA showed a significant effect of genotype (F(1,81) = 11.33; p = 0.0012) and treatment (F(5,81) = 11.42; p < 0.0001), as well as a significant interaction (F(5,81) = 5.14; p = 0.0004). One-way ANOVAs within genotype showed significant effects of treatment in both wild-type and D1-M4-KO mice (F(5,47) = 4.00; p = 0.005 and F(5,44) = 9.32; p < 0.0001, respectively). Post hoc tests showed that administration of amphetamine (2 mg/kg) induced a significant increase in distance moved when compared with vehicle in both wild-type (p < 0.05) and D1-M4-KO (p < 0.01) mice (Fig. 1). In wild-type mice, xanomeline dose dependently attenuated the amphetamine-induced hyperactivity, reaching significance at the 2 mg/kg dose (p < 0.05). In D1-M4-KO mice, xanomeline had no effect on the induced hyperactivity. Xanomeline (1 or 2 mg/kg) had no effect on baseline locomotor activity in either genotype. Bonferroni-corrected post hoc tests revealed a significant difference of the effect of xanomeline (2 mg/kg) between the two genotypes (p < 0.001).
Effects of xanomeline (1 and 2 mg/kg) on amphetamine (2 mg/kg)-induced locomotor activity in D1–M4-KO and wild-type mice. Data are presented as means ± SEM (n = 6–11). WT, Wild-type; KO, D1–M4-KO; veh, vehicle; xan, xanomeline; amph, amphetamine. *p < 0.05, **p < 0.01, and ###p < 0.001.
Reversal of apomorphine climbing by xanomeline is reduced in D1-M4-KO mice
In the first experiment, climbing behavior of D1-M4-KO mice and wild-type littermates was tested following treatment with vehicle or apomorphine (1 or 2 mg/kg) (Fig. 2). Overall two-way ANOVA showed a significant effect of genotype (F(1,54) = 12.26; p < 0.0009), treatment (F(2,54) = 61.96; p < 0.0001), and interaction (F(2,54) = 3.90; p < 0.0026). One-way ANOVA showed a significant effect of treatment with apomorphine on the climbing score in both wild-type (F(2,28) = 15.66; p < 0.0001) and D1-M4-KO (F(2,27) = 60.17; p < 0.0001) mice. In wild-type mice, post hoc analysis showed a significant and dose-dependent effect of apomorphine (1 and 2 mg/kg; p < 0.05 and p < 0.001, respectively) on climbing behavior. Also in D1-M4-KO mice, apomorphine induced climbing behavior at both doses (p < 0.001). Post hoc tests comparing the effect between genotypes found a significantly higher climbing score in D1-M4-KO mice following treatment with 1 mg/kg apomorphine (p < 0.001). Apomorphine at 2 mg/kg resulted in comparable climbing scores in both genotypes. In the next experiment, the attenuating effect of xanomeline on apomorphine (2 mg/kg)-induced climbing was investigated. Overall two-way ANOVA showed a significant effect of genotype (F(1,42) = 23.42; p < 0.0001) and treatment (F(2,42) = 15.26; p < 0.0001), and a near-significant interaction (F(2,42) = 3.19; p = 0.051). In wild-type animals, one-way ANOVA revealed a significant effect of xanomeline treatment on apomorphine-induced climbing (F(2,21) = 13.36; p = 0.0002) (Fig. 3). Compared with vehicle treatment, xanomeline (both 1 and 2 mg/kg) significantly attenuated climbing scores (p < 0.001). In D1-M4-KO mice, one-way ANOVA showed a significant effect of treatment (F(2,25) = 3.49; p < 0.05), but in contrast to wild-type animals, xanomeline was only able to significantly reduce climbing at the highest dose in the D1-M4-KO mice (p < 0.05). Bonferroni-corrected post hoc analysis showed a significant difference between genotypes at both xanomeline doses (p < 0.001 and p < 0.01, respectively).
Apomorphine-induced climbing in D1–M4-KO and wild-type mice. Data are given as means ± SEM (n = 8–12). *p < 0.05, ***p < 0.001 versus corresponding control group, and ###p < 0.001 versus same treatment group in wild-type controls.
Effects of xanomeline on apomorphine-induced (2 mg/kg) climbing in D1–M4-KO and wild-type controls. Data are given as means ± SEM (n = 7–9). *p < 0.05, ***p < 0.001 versus corresponding control group, and ###p < 0.001 versus same treatment group in wild-type controls.
Discussion
The results obtained in the present study show that the antipsychotic-like effects of xanomeline, investigated by amphetamine-induced hyperactivity and apomorphine-induced climbing in mice, were almost completely abolished in D1-M4-KO mice. The reported overall effects of xanomeline are in accordance with previous studies (Shannon et al., 2000; Woolley et al., 2009). We previously found that 2 mg/kg amphetamine induced a significantly larger increase in locomotor activity in D1-M4-KO mice relative to wild-type (Jeon et al., 2010). In the present study, D1-M4-KO mice similarly showed a trend toward an increased hyperlocomotor response to amphetamine compared with wild-type controls, although this did not reach significance. Methodological differences are likely responsible for this discrepancy. Interestingly, xanomeline had no effect on the amphetamine-induced hyperactivity in D1-M4-KO mice (Fig. 1), similar to findings obtained with whole-body M4 knock-out mice (Woolley et al., 2009).
D1-M4-KO mice displayed increased climbing behavior in response to apomorphine compared with wild-type mice (Fig. 2). This finding is in agreement with previous studies in these mice, where D1-M4-KO mice displayed a dopamine hyperresponsive phenotype with increased locomotor response to dopamine agonists, amphetamine-induced behavioral sensitization, and decreased response to antipsychotic-induced catalepsy (Jeon et al., 2010).
Shannon et al. (2000) found that xanomeline attenuated apomorphine climbing in mice at doses ≥3 mg/kg. In the present study, a 1 mg/kg dose was effective in wild-type mice. This is probably due to strain differences in responsiveness. More interestingly, the attenuating effect of xanomeline on apomorphine-induced climbing was significantly reduced in D1-M4-KO mice (Fig. 3). However, in contrast to the amphetamine-induced locomotor hyperactivity study, xanomeline was able to inhibit apomorphine-induced climbing at the highest dose in D1-M4-KO mice. Whether higher doses of xanomeline could inhibit amphetamine-induced hyperactivity in D1-M4-KO mice is unknown. However, higher xanomeline doses would probably affect baseline locomotor activity (Woolley et al., 2009).
The present findings extend our earlier data (Jeon et al., 2010) showing that M4 mAChRs present on D1 dopamine receptor-expressing neurons play an important role in the regulation of dopaminergic neurotransmission. D1 dopamine receptors activate, whereas M4 mAChRs inhibit, adenylyl cyclase and thereby regulate cAMP production through Gαs and Gαi/0 proteins, respectively (Onali and Olianas, 2002). The present findings therefore suggest that one mechanism by which xanomeline exerts its antipsychotic effect could be via M4 receptors, acting as functional D1 dopamine antagonists on the cAMP-dependent signaling pathways.
Since xanomeline is not entirely M1/M4-specific (Jakubík et al., 2006; Noetzel et al., 2009), classical cholinomimetic side effects, e.g., gastrointestinal side effects, were observed in humans (Bodick et al., 1997; Shekhar et al., 2008). Previous efforts to develop highly selective orthosteric agonists of the individual muscarinic subtypes have not been successful. However, selective positive allosteric modulators of the M4 mAChR have been developed recently (Brady et al., 2008; Chan et al., 2008), and in preclinical studies, positive allosteric modulation of M4 mAChR has been shown to attenuate apomorphine-induced deficits in prepulse inhibition (Chan et al., 2008) and amphetamine-induced hyperactivity in rats (Brady et al., 2008), supporting the potential role of the M4 mAChR as a future target for treatment of psychosis.
In conclusion, the antipsychotic-like effects of xanomeline investigated by amphetamine-induced hyperactivity and apomorphine-induced climbing were markedly attenuated in D1-M4-KO mice. This suggests that the specific subpopulation of M4 mAChRs localized on D1-containing neurons plays an important role in regulation of dopamine homeostasis and in the antipsychotic effects of xanomeline. Consequently, this study suggests a possible mechanism of action for the antipsychotic effects of xanomeline. The current data further support the hypothesis that activation of the M4 mAChR represents a potential target for the medical treatment of psychosis.
Footnotes
This work was supported by the Ivan Nielsen Foundation, the Lundbeck Foundation, and the Aase and Ejnar Danielsen Foundation.
The authors declare no competing financial interests.
- Correspondence should be addressed to Anders Fink-Jensen, Laboratory of Neuropsychiatry, Psychiatric Centre Copenhagen, University of Copenhagen, Denmark, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark. a.fink-jensen{at}dadlnet.dk
