The Natural Protoalkaloid Methyl-2-Amino-3-Methoxybenzoate (MAM) Alleviates Positive as well as Cognitive Symptoms in Rat and Mouse Schizophrenia Models

Abstract

The development of new antipsychotics with pro-cognitive properties and less side effects represents a priority in schizophrenia drug research. In this study, we present for the first time a preclinical exploration of the effects of the promising natural atypical antipsychotic Methyl-2-Amino-3- Methoxybenzoate (MAM), a brain-penetrable protoalkaloid from the seed of the plant Nigella damascena. Using animal models related to hyperdopaminergic activity, namely the pharmacogenetic apomorphine (D2/D1 receptor agonist)-susceptible (APO-SUS) rat model and pharmacologically induced mouse and rat models of schizophrenia, we found that MAM reduced gnawing stereotypy and climbing behaviours induced by dopaminergic agents. This predicts antipsychotic activity. In line, MAM antagonized apomorphine-induced c-Fos and NPAS4 mRNA levels in post-mortem brain nucleus accumbens and dorsolateral striatum of APO-SUS rats. Furthermore, phencyclidine (PCP, an NMDA receptor antagonist) and 2,5-Dimethoxy-4-iodoamphetamine (DOI, a 5HT2A/2C receptor agonist) induced prepulse inhibition deficits, reflecting the positive symptoms of schizophrenia, which were rescued by treatment with MAM and atypical antipsychotics alike. Post-mortem brain immunostaining revealed that MAM blocked the strong activation of both PCP- and DOI-induced c-Fos immunoreactivity in a number of cortical areas. Finally, during a 28-day subchronic treatment regime, MAM did not induce weight gain, hyperglycemia, hyperlipidemia or hepato- and nephrotoxic effects, side effects known to be induced by atypical antipsychotics. MAM also did not show any cataleptic effects. In conclusion, its brain penetrability, the apparent absence of preclinical side effects, and its ability to antagonize positive and cognitive symptoms associated with schizophrenia make MAM an exciting new antipsychotic drug that deserves clinical testing.

Keywords: APO-SUS; MAM; PPI; antipsychotics; catalepsy; gnawing; procognitive.; schizophrenia.

Fig. (1)

Effects of acute subcutaneous MAM (3, 10, 30 mg/kg) and clozapine (10 mg/kg) pretreatments on the gnawing stereotypy induced by apomorphine (1.5 mg/kg) in APO-SUS rats. (A). Schematic representation of the experimental paradigm. (B). Gnawing counts in APOSUS rats treated with VEH+SAL (control group), VEH + APO (positive control group), CLOZ (10 mg/kg) + APO (standard group), or MAM (3, 10, 30 mg/kg) + APO. All pretreatments were injected subcutaneously 15 min before APO injection into the neck (n = 9-10 rats per group). (C). Gnawing counts in APO-UNSUS compared to APO-SUS rats (independent samples t-test). Values represent average stereotypy gnawing counts every 3 min±SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 (comparison vs. control group/between group’s comparison); ^####P < 0.0001 (comparison vs. apomorphine-treated group), using one-way ANOVA, with Dunnett's test, followed by post-hoc Tukey's test. (D) Comparative gnawing counts over time during 45 min session in APOSUS rats pre-treated with the most effective dose of MAM (10 mg/kg), CLOZ or VEH, before apomorphine. (A higher resolution/colour version of this figure is available in the electronic copy of the article).

Fig. (2)

Effects of acute subcutaneous MAM (3, 10, and 30 mg/kg) and clozapine (10 mg/kg) pretreatments on the startle reactivity and prepulse inhibition (PPI) deficits induced by PCP (1.5 mg/kg) or DOI (3 mg/kg) in Wistar rats (A). Schematic representation of the experimental paradigm (C, E). Effects of MAM and CLOZ pretreatment on startle reactivity induced by PCP and DOI (B, D). Effects of MAM and CLOZ pretreatment on the percentage change of PPI deficits induced by PCP or DOI at different prepulse intensities: 3, 5, and 10dB above the background noise (PP3, PP5, and PP10, respectively). Values represent the average startle in Vmax (C, E) or average PPI% (B, D) ± SEM. Oneway ANOVA, with Dunnett's test, followed by post-hoc Tukey's test, was used for comparisons: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (comparison vs. control group); ^#P < 0.05 (comparison vs. DOI vs. PCP treated group). The male Wistar rats used for both tests are different and naive to both treatments (n = 9-10 rats per group used for PCP and n = 10-11 for DOI). The average PPI (%) ± SEM was calculated as the average responses to all of the pulse-alone or prepulse trials. (A higher resolution/colour version of this figure is available in the electronic copy of the article).

Fig. (3)

Effects of MAM on spatial working memory deficits in APO-SUS rats (T-maze test) (A). Schematic representation of the experimental paradigm (C, E). Spatial working memory deficit presented in APO-SUS compared to APO-UNSUS rats treated with vehicle and their respective latencies (Independent t-tests, n=8) (B, D). The effects of vehicle, MAM (3, 10, 30 mg/kg), and CLOZ (10 mg/kg) on spatial working memory deficits presented in APO-SUS rats and their respective latencies (ANOVA, n=8) (F). Effect of MAM (10 mg/kg) and vehicle on APO-SUS reversal learning during 3 consecutive days. Values represent the average percentage of correct trials or their latencies to retrieve food reward pellets (±SEM) in 3 daily consecutive trials (30 in total) of the spontaneous delayed alternation. One-way ANOVA, with Dunnett's test, followed by post-hoc Tukey's test was used for comparisons: **P < 0.01, ***P < 0.001 (comparison vs. control group), ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 (comparison vs. CLOZ treated group).

Fig. (4)

Effects of acute subcutaneous MAM (10, 30 mg/kg) and clozapine (10 mg/kg) pretreatments on c-Fos and NPAS4 mRNA levels induced by apomorphine (1.5 mg/kg) in APO-SUS rats. (B, D, F, H). c-Fos and NPAS4 mRNA levels induced by apomorphine in the nucleus accumbens and dorsolateral striatum in APO-UNSUS compared to APO-SUS rats (independent samples t-tests). (A, C, E, G). APO-SUS rats treated with VEH+SAL (control group), VEH+APO (positive control group), CLOZ (10 mg/kg)+APO (standard group), or MAM (10, 30 mg/kg)+APO. Values represent the average of %mRNA fold vs. control ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001 (comparison vs. control group/between group’s comparison); ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 (comparison vs. apomorphine-treated group), using one-way ANOVA, with Dunnett's test, followed by post-hoc Tukey's test. All pretreatments were injected subcutaneously 15 min before APO injection into the neck (n = 9-10), and rats were killed immediately after the gnawing box test.

Fig. (5)

Effects of pretreatment of MAM (3, 10, 30 mg/kg) and clozapine (10 mg/kg) on the c-Fos immunoreactivity induced by DOI (3 mg/kg) in Wistar rats. (A) In each area, c-Fos immunoreactive cell counts are present (average ± SEM) in rat cortical areas induced by vehicle+saline or DOI (3 mg/kg) pretreated with vehicle, CLOZ (10 mg/kg), and MAM (3, 10, 30 mg/kg). (B) Representative images from each area and treatment. One-way ANOVA, with Dunnett's test, followed by post-hoc Tukey's test, was used for comparisons: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (comparison vs. control group/between group’s comparison), ^###P < 0.001, ^####P < 0.0001 (comparison vs. DOI-treated group), in each area. The average number of c-Fos positive cells/region of interest ± SEM (4-5 slices per rat, n=8 rats per group) was calculated using Image J software.

Fig. (6)

Effects of pretreatment of MAM (3, 10, and 30 mg/kg) and clozapine (10 mg/kg) on the c-Fos immunoreactivity induced by PCP (1.5 mg/kg) in Wistar rats. (A) In each area, c-Fos immunoreactive cell counts are present (average ± SEM) in rat cortical areas induced by (VEH+SAL) or PCP (1.5 mg/kg) pretreated with vehicle, CLOZ (10 mg/kg), and MAM (3, 10, 30 mg/kg). (B) Representative images from each area and treatment. One-way ANOVA, with Dunnett's test, followed by post-hoc Tukey's test, was used for comparisons: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (comparison vs. control group/between group’s comparison), ^#P < 0.05, ^###P < 0.001, ^####P < 0.0001 (comparison vs. CLOZ treated group), in each area. The average number of c-Fos positive cells/region of interest ± SEM (4-5 slices per rat, n=8 rats per group) was calculated using Image J software.