An ontogenic study of receptor mechanisms by which acute administration of low-doses of methamphetamine suppresses DOI-induced 5-HT2A-receptor mediated head-twitch response in mice

Abstract

Background: Methamphetamine (MA) is a non-selective monoamine releaser and thus releases serotonin (5-HT), norepinephrine (NE) and dopamine (DA) from corresponding nerve terminals into synapses. DOI ((±)-2, 5-dimethoxy-4-iodoamphetamine) is a direct-acting serotonergic 5-HT_2A/C receptor agonist and induces the head-twitch response (HTR) via stimulation of 5-HT_2A receptor in mice. While more selective serotonin releasers such as d-fenfluramine evoke the HTR, monoamine reuptake blockers (e.g., cocaine) suppress the DOI-evoked HTR via indirect stimulation of serotonergic 5-HT_1A- and adrenergic ɑ₂-receptors. Since the induction of HTR by DOI is age-dependent, we investigated whether: (1) during development MA can evoke the HTR by itself, and (2) acute pretreatment with either the selective 5-HT_2A receptor antagonist EMD 281014 or low-doses of MA can: (i) modulate the DOI-induced HTR in mice across postnatal days 20, 30 and 60, and (ii) alter the DOI-induced c-fos expression in mice prefrontal cortex (PFC). To further explore the possible modulatory effect of MA on DOI-induced HTR, we investigated whether blockade of inhibitory serotonergic 5-HT_1A- or adrenergic ɑ₂-receptors by corresponding selective antagonists (WAY 100635 or RS 79948, respectively), can prevent the effect of MA on DOI-induced HTR during aging.

Results: Although neither EMD 281014 nor MA by themselves could evoke the HTR, acute pretreatment with either EMD 281014 (0.01, 0.05 and 0.1 mg/kg, i.p.) or MA (1, 2.5, 5 mg/kg, i.p.), dose-dependently suppressed the DOI-induced HTR across ages. While WAY 100635 significantly reversed the inhibitory effect of MA in 20- and 30-day old mice, RS 79948 failed to significantly counter MA's inhibitory effect. Moreover, DOI significantly increased c-fos expressions in several PFC regions. EMD 281014 prevented the DOI-induced increases in c-fos expression. Despite the inhibitory effect of MA on DOI-induced HTR, MA alone or in combination with DOI, significantly increased c-fos expression in several regions of the PFC.

Conclusion: The suppressive effect of MA on the DOI-evoked HTR appears to be mainly due to functional interactions between the HTR-inducing 5-HT_2A receptor and the inhibitory 5-HT_1A receptor. The MA-induced increase in c-fos expression in different PFC regions may be due to MA-evoked increases in synaptic concentrations of 5-HT, NE and/or DA.

Keywords: 5-HT1A receptor; 5-HT2A receptor; DOI; Head-twitch response; Methamphetamine; ɑ2-adrenergic receptor.

Fig. 1

Timelines for injections, HTR observation, and perfusion. a and b Corresponding vehicle (i.p.), or varying doses of the EMD 281014 (0.01, 0.05, 0.1 mg/kg, i.p.), or MA (1, 2.5, 5 mg/kg, i.p.), were injected to different groups of mice (20-, 30- or 60-day old) 30 min prior to DOI (1 mg/kg, i.p.) injection. The frequency of HTRs were observed for 30 min post DOI injection. Based on behavioral data, 30-day-old mice were selected to perform the immuno-histochemistry studies. Thus, after injections and HTR observation, mice were perfused at 120 min (a, b). For behavioral interaction studies, different groups of mice (20-, 30, and 60-day old) received either a single dose of MA (5 mg/kg, i.p.) or its vehicle at 0 min, and at 20 min received either the 5-HT_1A receptor antagonist WAY 100635 (0.25 mg/kg, i.p.), or the ɑ₂-adrenergic-receptor antagonist RS 79948 (0.1 mg/kg, i.p.), or corresponding vehicle. At 30 min DOI (1 mg/kg, i.p.) was injected (c, d) and the HTR frequency were observed for 30 min post DOI injection

Fig. 2

a−e show different regions of the PFC sections of the vehicle + DOI treated mice brain at 5 coronal levels (− 2.68 mm, − 2.34 mm, − 2.1 mm, − 1.9 mm, and − 1.7 mm relative to bregma Paxinos and Franklin [38]), in which c-fos numbers were counted, scale bars = 500 μm (a−e). Illustrations are adapted from the atlas of Paxinos and Franklin [38]. c-fos immunoreactivity was counted when the cell nucleus was round or oval, completely filled and double-labeled with DAPI, scale bars = 10 μm (f−h). Abbreviations: AI: agranular insular cortex; Cg1: cingulate cortex area 1; DLO: dorsal lateral orbital cortex; DP: dorsal peduncular cortex; FrA: frontal associated cortex; IL: infralimbic cortex; LO: lateral orbital cortex; M1: primary motor cortex; M2: secondary motor cortex; MO: medial orbital cortex; PrL: prelimbic cortex, S1: primary somatosensory area; VO: ventral orbital cortex

Fig. 3

Suppressive effects of varying doses of the selective 5-HT_2A receptor antagonist EMD 281014 (0, 0.01, 0.05 and 0.1 mg/kg, i.p.) on the frequency of HTR induced by DOI (1 mg/kg, i.p.) across different ages (20-, 30- and 60-day old) in mice. In EMD 281014 vehicle-pretreated control mice, the mean frequency of DOI-induced HTR tended to gradually decrease with increasing age, with significant differences occurring between 20- and 60-day (^####p < 0.0001) and between 30- and 60-day old mice (^###p = 0.0002). Varying doses of EMD 281014 suppressed DOI-induced HTR in a dose- and age-dependent manner across different ages. Compared to corresponding age-matched vehicle-pretreated control group, significant reductions were observed at all tested doses of EMD 281014 in 20- and 30-day old mice, but only at 0.05 and 0.1 mg/kg doses in 60-day old mice. *p = 0.0181, ****p < 0.0001 vs. vehicle injection; two-way ANOVA followed by Bonferroni's test. n = 6 in each group. Data are presented as means ± SEM.

Fig. 4

Suppressive effects of varying doses of MA (0, 1, 2.5 5 mg/kg, i.p.) on the frequency of HTR induced by DOI (1 mg/kg, i.p.) across different ages (20-, 30- and 60-day old) in mice. In the MA vehicle-pretreated control group, the frequencies of DOI-induced HTR gradually decreased with increasing age. Significant differences were found between 20- and 60-day (^####p < 0.0001), and between 30- and 60-day old mice (^####p < 0.0001). Relative to the corresponding age-matched vehicle-pretreated controls, MA inhibited the mean frequency of DOI-induced HTRs across different ages in a dose- and age-dependent manner. Significant differences were observed at all tested doses of MA in 20- and 30-day old mice, whereas the significant effect was only observed at 5 mg/kg of MA in 60-day old mice. *p = 0.0432, ****p < 0.0001 vs. vehicle injection; two-way ANOVA followed by Bonferroni's test. n = 5−8 in each group. Data are presented as means ± SEM.

Fig. 5

Reversal effect of the selective 5-HT_1A receptor antagonist WAY 100635 (0.25 mg/kg, i.p.) on the inhibitory action of MA (5 mg/kg, i.p.) on DOI-induced (1 mg/kg, i.p.) HTR across different ages (20-, 30-, and 60-day old) in mice. Relative to the corresponding age-matched Vehicle + Vehicle + DOI pretreated control group, MA pretreatment (i.e. MA + Vehicle + DOI treatment group) significantly reduced the frequency of DOI-induced HTR in 20- 30- (all ****p < 0.0001) and 60-day old mice (*p = 0.0404). WAY 100635 by itself (i.e. in the Vehicle + WAY 100635+ DOI group) markedly attenuated the frequency of DOI-induced HTR in 20-day mice but did not affect the evoked behavior in 30- or 60-day old mice. Relative to the corresponding MA + Vehicle + DOI treatment control group at each age group, inclusion of the 5-HT_1A receptor antagonist WAY 100635 (i.e. the MA + WAY 100635 + DOI treatment group) reversed the inhibitory effect of MA on DOI-induced HTR across all ages but significance was observed only during days 20 and 30. ^#p = 0.0239, ^####p < 0.0001 vs. MA + Vehicle + DOI group; two-way ANOVA followed by Bonferroni's test. n = 6 − 10 in each group. Data are presented as means ± SEM.

Fig. 6

Effects of the selective ɑ₂-adrenergic receptor antagonist RS 79948 (0.25 mg/kg, i.p.) on the inhibitory action of MA (5 mg/kg, i.p.) on the frequency of HTR induced by DOI (1 mg/kg, i.p.) across different ages (20-, 30- and 60-day old) in mice. Compared to the corresponding Vehicle + Vehicle + DOI treatment control group at each age group, inclusion of MA (i.e. MA + Vehicle + DOI treatment group) significantly decreased the mean frequency of HTR induced by DOI in 20- and 30-day old mice, but no significant effect was seen in 60-day old mice. RS 79948 pretreatment tended to reverse the inhibitory effect of MA (i.e. MA + RS 79948 + DOI treatment group) across different ages but the effect failed to attain significance. ***p = 0.001, ****p < 0.0001 vs. Vehicle + Vehicle + DOI group. n.s.: no significant difference vs. MA + Vehicle + DOI group; two-way ANOVA followed by Bonferroni's test. n = 5 − 10 in each group. Data are presented as means ± SEM.

Fig. 7

Suppressive effects of the selective 5-HT_2A receptor antagonist EMD 281014 on DOI-induced c-fos expression in different regions at 5 coronal sections in the PFC. Compared to the corresponding vehicle + vehicle pretreated control-mice, administration of DOI (i.e. vehicle + DOI (1 mg/kg, i.p.)) evoked greater numbers of c-fos positive cells in the: (i) FrA, LO, and DLO at the level of − 2.68 mm relative to bregma (a, b); (ii) M1, M2, LO, and AI at the level of − 2.34 mm relative to bregma (c, d); (iii) M1, LO, and AI at the level of − 2.1 mm relative to bregma (e, f); (iv) S1, M1, IL, and AI at the level of − 1.98 mm relative to bregma (g, h) and (v) S1 and M1 at the level of − 1.7 mm relative to bregma (i, j). Pretreatment with the selective 5-HT_2A receptor antagonist EMD 281014 (0.1 mg/kg, i.p.) prevented the DOI-induced c-fos expression in the: (i) FrA, MO, LO, and DLO at the level of − 2.68 mm relative to bregma (a, b); (ii) M1, M2, LO, and AI at the level of − 2.34 mm relative to bregma (c, d); (iii) M1, M2, LO, and AI at the level of − 2.1 mm relative to bregma (e, f); (iv) S1, M1, IL, VO, and AI at the level of − 1.98 mm relative to bregma (g, h); and (v) S1 and M1 at the level of − 1.7 mm relative to bregma (i, j). Treatment with EMD 281014 by itself (i.e. EMD 281014 + vehicle group) did not produce a change in c-fos expression in any of the regions tested when compared to vehicle + vehicle treatment control group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Vehicle + Vehicle pretreated control-mice. ^#p < 0.05, ^##p < 0.01 ^###p < 0.001 vs. Vehicle + DOI treatment group one-way ANOVA followed by Tukey's test. Data are presented as means ± SEM

Fig. 8

Effects of DOI and MA administration either alone or in combination on c-fos expression in different regions at 5 coronal levels in the PFC. Relative to the corresponding vehicle + vehicle treatment control group, DOI by itself (i.e. vehicle + DOI (1 mg/kg, i.p.)) significantly increased c-fos expression in the: (i) FrA, LO, and DLO at the level of − 2.68 mm relative to bregma (a, b); (ii) M1, M2, LO, and AI at the level of − 2.34 mm relative to bregma (c, d); (iii) M1, LO, and AI at the level of − 2.1 mm relative to bregma (e, f); (iv) S1, M1, PrL, IL, and AI at the level of − 1.98 mm relative to bregma (g, h) and (v) S1 and M1 at the level of − 1.7 mm relative to bregma (i, j). MA by itself (i.e. MA + vehicle group) significantly increased the expressions of c-fos in the: (i) PrL, VO, LO, and DLO at the level of − 2.68 mm relative to bregma (a, b); (ii) M2, Cg1, VO, and AI at the level of − 2.34 mm relative to bregma (c, d); (iii) M2, and AI at the level of − 2.1 mm relative to bregma (e, f). (iv) M2, Cg1, PrL, and AI at the level of − 1.98 mm relative to bregma (g, h). Combined treatment with MA + DOI significantly increased the expressions of c-fos in the: (i) FrA, PrL, LO, and DLO at the level of − 2.68 mm relative to bregma (a, b); (ii) M1, M2, and AI at the level of − 2.34 mm relative to bregma (c, d); (iii) M1, and AI at the level of − 2.1 mm relative to bregma (e, f); (iv) S1, Cg1, PrL and AI at the level of − 1.98 mm relative to bregma (g, h); and (v) S1 at the level of − 1.7 mm relative to bregma (i, j). There are no significant differences among comparisons of vehicle + DOI vs. MA + vehicle; vehicle + DOI vs. MA + DOI; and MA + vehicle vs. MA + DOI in the c-fos expressions in these brain regions. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Vehicle + Vehicle treatment control group; one-way ANOVA followed by Tukey's test. Data are presented as means ± SEM