Impact of serotonin 2C receptor null mutation on physiology and behavior associated with nigrostriatal dopamine pathway function

Abstract

The impact of serotonergic neurotransmission on brain dopaminergic pathways has substantial relevance to many neuropsychiatric disorders. A particularly prominent role has been ascribed to the inhibitory effects of serotonin 2C receptor (5-HT(2C)R) activation on physiology and behavior mediated by the mesolimbic dopaminergic pathway, particularly in the terminal region of the nucleus accumbens. The influence of this receptor subtype on functions mediated by the nigrostriatal dopaminergic pathway is less clear. Here we report that a null mutation eliminating expression of 5-HT(2C)Rs produces marked alterations in the activity and functional output of this pathway. 5-HT(2C)R mutant mice displayed increased activity of substantia nigra pars compacta (SNc) dopaminergic neurons, elevated baseline extracellular dopamine concentrations in the dorsal striatum (DSt), alterations in grooming behavior, and enhanced sensitivity to the stereotypic behavioral effects of d-amphetamine and GBR 12909. These psychostimulant responses occurred in the absence of phenotypic differences in drug-induced extracellular dopamine concentration, suggesting a phenotypic alteration in behavioral responses to released dopamine. This was further suggested by enhanced behavioral responses of mutant mice to the D(1) receptor agonist SKF 81297. Differences in DSt D(1) or D(2) receptor expression were not found, nor were differences in medium spiny neuron firing patterns or intrinsic membrane properties following dopamine stimulation. We conclude that 5-HT(2C)Rs regulate nigrostriatal dopaminergic activity and function both at SNc dopaminergic neurons and at a locus downstream of the DSt.

Figure 1.

5-HT_2CR mutant mice display increased SNc dopaminergic neuronal activity. A, Numbers of spontaneously active dopaminergic neurons encountered while recording from SNc and VTA revealed no phenotypic difference. B, 5-HT_2CR mutant mice exhibit increased dopaminergic cell firing in SNc but not in VTA (5.38 ± 0.23 vs 4.49 ± 0.22 spikes/s for SNc, p < 0.01; 4.55 ± 0.21 vs 4.60 ± 0.16 spikes/s for VTA, NS). C, 5-HT_2CR mutant mice exhibit increased dopaminergic cell burst firing in SNc but not VTA (16.2 ± 2.5% vs 8.6 ± 1.9% cells bursting for SNc, p < 0.05; 16.6 ± 2.5% vs 17.5 ± 2% cells bursting for VTA, NS). D, E, Interspike interval histograms for SNc neurons recorded from wild-type (D) and 5-HT_2CR mutant (E) mice demonstrate that SNc neurons from mutant mice have overall higher-frequency activity spike trains than those seen in wild-type mice. Data are represented as mean ± SEM. n = 8 per genotype. *p < 0.05; **p < 0.01.

Figure 2.

5-HT_2CR mutant mice display elevated striatal extracellular dopamine levels. A, B, No net flux plots in DSt (A) and NAc (B) of DA from 5-HT_2CR mutant and wild-type mice. The y-axis indicates DA concentration differences between aCSF entering the probe (C_in) and aCSF exiting the probe (C_out). The point of no net flux (y = 0 nm; horizontal dotted line) corresponds to DA equilibrium between tissue and microdialysis probe revealing extracellular DA concentration (arrows). Estimates of in vivo DA recovery in the DSt, as indicated by the slope of the no net flux curve, did not differ between 5-HT_2CR mutant mice (0.32 ± 0.03) and wild types (0.45 ± 0.07, F_(1,14) = 3.537, NS). Similarly, estimates of in vivo DA recovery in the NAc did not differ between groups (5-HT_2CR mutant mice, 0.29 ± 0.04; wild types, 0.34 ± 0.03, F_(1,14) = 0.881, NS). C, 5-HT_2CR mutant mice display elevated mean basal dopamine concentration in the DSt (17 ± 2.1 nm vs 9.6 ± 1.6 nm in wild types, F_(1,15) = 8.924, p < 0.01) and NAc (8.5 ± 0.7 nm vs 6.3 ± 0.8 nm in wild types, F_(1,15) = 4.887, p < 0.05). Data are represented as mean ± SEM. n = 8 per genotype. *p < 0.05; **p < 0.01.

Figure 3.

5-HT_2CR mutant mice exhibit altered syntactic grooming chains. A, Increased duration of phase 3 and decreased duration of phase 4 syntactic grooming in 5-HT_2CR mutant mice. Mean phase 3 duration was 29.8 ± 2.2 s in wild-type mice, 39.8 ± 5 s in 5-HT_2CR mutant mice (p < 0.05); mean phase 4 duration was 239.2 ± 17.9 s in wild-type mice, 192.0 ± 14.2 s in 5-HT_2CR mutant mice (p < 0.03). B, Alteration of nonsyntactic grooming properties in 5-HT_2CR mutant mice. 5-HT_2CR mutant mice exhibit increased nonsyntactic grooming event initiation (5.20 ± 0.4 grooming events per minute vs 4.07 ± 0.16 in wild types; p < 0.015) and reduced nonsyntactic grooming duration (1.89 ± 0.1 vs 2.55 ± 0.11 s in wild types, p < 0.0007). Data are represented as mean ± SEM. n = 8 per genotype. *p < 0.05; **p < 0.001.

Figure 4.

5-HT_2CR mutant mice display altered motor responses to d-amphetamine. ○, d-Amphetamine-treated wild-type mice; ●, d-amphetamine-treated 5-HT_2CR mutant mice; □, saline-treated wild-type mice; ■, saline-treated 5-HT_2CR mutant mice. A, Locomotor activity following 2.5 mg/kg d-amphetamine. Repeated-measures ANOVA on locomotor distance traveled between 0 and 90 min after injection revealed significant effects of treatment (F_(1,27) = 131.14; p < 0.001) and treatment × time interaction (F_(1,27) = 11.69; p < 0.01). Inset, ANOVA conducted on peak activity showed significant effects of treatment (F_(1,26) = 175.69; p < 0.001) and treatment × genotype interaction (F_(1,26) = 5.79; p < 0.05). B, Locomotor activity following 5 mg/kg d-amphetamine. Repeated-measures ANOVA on locomotor distance traveled between 0 and 90 min after injection revealed significant effects of time (F_(1,26) = 9.24; p < 0.01), treatment (F_(1,26) = 301.19; p < 0.001), and treatment × genotype interaction (F_(1,26) = 6.94; p < 0.05). Inset, ANOVA conducted on peak activity showed a significant effect of treatment (F_(1,26) = 196.26; p < 0.001). C, Locomotor activity following 10 mg/kg d-amphetamine. Repeated-measures ANOVA on locomotor distance traveled between 0 and 90 min after injection showed significant effects of genotype (F_(1,26) = 6.04, p < 0.05), treatment (F_(1,26) = 41.31, p < 0.001), and time (F_(1,26) = 215.61, p < 0.001), as well as interactions of time × genotype (F_(1,26) = 4.93; p < 0.05), treatment × genotype (F_(1,26) = 12.85; p < 0.01), and time × treatment (F_(1,26) = 47.82; p < 0.001). The tertiary interaction of time × treatment × genotype was also significant (F_(1,26) = 10.25; p < 0.01). Inset, ANOVA conducted on peak activity showed significant effects of treatment (F_(1,26) = 63.44; p < 0.001) and treatment × genotype (F_(1,26) = 5.76; p < 0.05). D, Repeated-measures ANOVA conducted on percentage time spent in stereotypy following 2.5 mg/kg d-amphetamine revealed no significant phenotypic differences. E, 5-HT_2CR mutant mice display greater focused stereotypy following 5 mg/kg d-amphetamine (F_(1,11) = 5.14, p < 0.05). F, No phenotypic difference in focused stereotypy following 10 mg/kg d-amphetamine. Data are represented as mean ± SEM. n = 7–8 per genotype per treatment.

Figure 5.

Enhanced sensitivity of 5-HT_2CR mutant mice to induction of motor stereotypy by dopamine reuptake blockade. A, Increased GBR 12909-evoked stereotypy observed in 5-HT_2CR mutant mice compared with wild types (F_(3,56) = 4.017, p < 0.012 for genotype × dose interaction). Behavior evaluated 90 min after drug administration. B, Increased GBR 12909-evoked stereotypy observed in C57BL/6J mice following treatment with the 5-HT_2CR inverse agonist SB 206553 (F_(3,55) = 3.764, p < 0.016 for SB 206553 × GBR 12909 dose interaction). In animals receiving both GBR 12909 and SB 206553, drugs were administered simultaneously. Behavior evaluated 90 min after drug administration. Data are represented as mean ± SEM. n = 7 per genotype per treatment.

Figure 6.

No phenotypic effect on DSt extracellular dopamine concentrations after treatment with d-amphetamine or GBR 12909. ○, Wild-type mice; ●, 5-HT_2CR mutant mice. A–D, DSt extracellular dopamine following d-amphetamine 2.5 mg/kg (A), d-amphetamine 5 mg/kg (B), GBR 12909 3 mg/kg (C), and GBR 12909 10 mg/kg (D). For A–D, no phenotypic differences were noted between drug- and vehicle-treated groups. Arrows indicate the time of drug administration. Data are represented as mean ± SEM. n = 5–8 per genotype per treatment.

Figure 7.

5-HT_2CR mutant mice display decreased locomotion in response to systemic treatment with the D₁R agonist SKF 81297. Repeated-measures ANOVA on locomotor distance traveled between 0 and 90 min after injection revealed significant effects of time (F_(1,25) = 39.1, p < 0.001) and time × genotype (F_(1,25) = 4.65, p < 0.05) and time × treatment (F_(2,25) = 5.89, p < 0.01) interactions. There was also a significant time × genotype × treatment interaction (F_(2,25) = 10.69, p < 0.001). ANOVA conducted on peak activity showed significant effects of genotype (F_(1,25) = 23.49, p < 0.001), treatment (F_(2,25) = 60.46, p < 0.001), and genotype × treatment interaction (F_(2,25) = 22.06, p < 0.001). Data are represented as mean ± SEM. n = 6–7 per group.