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.2018 Aug 14:12:173.
doi: 10.3389/fnbeh.2018.00173. eCollection 2018.

Behavioral Effects of Acute Systemic Low-Dose Clozapine in Wild-Type Rats: Implications for the Use of DREADDs in Behavioral Neuroscience

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Behavioral Effects of Acute Systemic Low-Dose Clozapine in Wild-Type Rats: Implications for the Use of DREADDs in Behavioral Neuroscience

Ann-Kathrin Ilg et al. Front Behav Neurosci..

Abstract

Designer receptors exclusively activated by designer drugs (DREADDs) are popular tools used to manipulate the activity of defined groups of neurons. Recent work has shown that DREADD effects in the brain are most likely not mediated by the proposed ligand clozapine-N-oxide (CNO) but its metabolite clozapine (CLOZ). However, it is not known whether low doses of CLOZ required to activate DREADDs already have DREADD-independent effects on behavior as described for higher CLOZ doses used in previous preclinical studies. To close this gap, we compared effects of acute systemic (i.p.) CLOZ treatment vs. vehicle (VEH) in a wide range of behavioral tests in male wild-type rats. We found that CLOZ doses as low as 0.05-0.1 mg/kg significantly affected locomotion, anxiety and cognitive flexibility but had no effect on working memory or social interaction. These results highlight the need for careful controls in future chemogenetic experiments and show that previous results in studies lacking CNO/CLOZ controls may require critical re-evaluation.

Keywords: CNO; clozapine; designer receptors exclusively activated by designer drugs (DREADD); elevated plus-maze; locomotion; rats; set-shifting; social interaction.

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Figure 1
Figure 1
Clozapine/CLOZ reduces locomotor activity and induces an anxious phenotype but does not affect social interaction.(A) Locomotor activity: directly after placing rats (n = 19) in the open field, they showed high locomotor activity and reached a steady state after 15 min (data of the second vehicle injection/VEH 2 and saline/SAL were not different from VEH and are not shown to improve readability of the figure).(B) Track length in all conditions is expressed as percentage of the VEH 1 baseline value. For this steady-state activity, track length was affected by subject (repeated-measures one-way ANOVA followed by Dunnett’s test;F(18,90) = 8.85;p = 3 × 10−13) and treatment (F(5,90) = 16.34;p = 2 × 10−11). We observed a dose-dependent decrease after low-dose CLOZ administration (all compared against VEH injection). The lowest doses used caused similar decreases in track length (0.1 mg/kg CLOZ:p = 2 × 10−4, this corresponds to 69.2 ± 7.5% of the track length after VEH injection; 0.05 mg/kg CLOZ:p = 10−4; 72.1 ± 8.73%). There was an even stronger reduction with 1 mg/kg CLOZ (p < 10−4; 40.9 ± 5.7%). The second vehicle (VEH 2) injection was performed to test if repetition influences steady-state activity, but no difference was observed (p = 0.27; 95.4 ± 10.2%). Also, SAL treatment didn’t change the track length (p = 0.56; 101.1 ± 10.2%). Data are represented as mean ± standard error of the mean (SEM).(C,D) Elevated plus-maze: the percentage of open arm entries/%OAE (H = 13.0,p = 0.0046, Kruskal-Wallis test) and the percentage of open arm time/%OAT (H = 11.3,p = 0.010, Kruskal-Wallis test) was different between groups.Post hoc tests showed that the dose of 0.1 mg/kg induced an anxious phenotype. Both the %OAE (p = 0.023, Dunn’s multiple comparisons test) and %OAT (p = 0.023) were decreased with this dose (n = 9). 0.05 mg/kg and 1 mg/kg CLOZ did not affect the %OAE or the %OAT significantly. The total number of entries (controlling for locomotor activity effects) were not different between groups (p = 0.24, Kruskal-Wallis test).(E) Social interaction: no significant differences concerning cumulative active social interaction time were observed with both doses tested (0.1 mg/kg and 0.3 mg/kg CLOZ,p = 0.99 andp = 0.57,n = 10 per group). Data displayed as median and the first and third quartile (P25, P75; Tukey-style whiskers) *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 2
Figure 2
CLOZ increases cognitive flexibility but does not affect working memory performance.(A,B) Strategy set-shifting: during the visual baseline, rats (n = 7 per group) did not differ with respect to performance, whereas they reached the criterion during the shift from the visual to the place rule in fewer trials (CLOZ 0.1 mg/kg vs. VEH;p = 0.011). Trials to criterion were not different for the shift back to visual rule. The better performance during the shift to the place rule went along with a lower number of old errors (i.e., responses that are correct according the previous rule;p = 0.016). This was caused by a decrease in the number of regressive (p = 0.016) but not perseverative errors, indicating that CLOZ improves rule maintenance after the switch. The number of never-reinforced errors was not different. Data shown as median (P25, P75). *p < 0.05.(C) Delayed alternation task: performance decreased significantly when longer delays separated two consecutive choice trials (F(4,72) = 9.42;p = 4 × 10−6), but there was no difference between VEH or CLOZ treatment (F(1,18) = 0.93;p = 0.347;n = 19 rats). Data are expressed as mean ± SEM.
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