Local modulation of striatal glutamate efflux by serotonin 1A receptor stimulation in dyskinetic, hemiparkinsonian rats

Abstract

Serotonin 1A receptor (5-HT(1A)R) agonists reduce both L-DOPA- and D1 receptor (D1R) agonist-mediated dyskinesia, but their anti-dyskinetic mechanism of action is not fully understood. Given that 5-HT(1A)R stimulation reduces glutamatergic neurotransmission in the dopamine-depleted striatum, 5-HT(1A)R agonists may diminish dyskinesia in part through modulation of pro-dyskinetic striatal glutamate levels. To test this, rats with unilateral medial forebrain bundle dopamine or sham lesions were primed with L-DOPA (12 mg/kg+benserazide, 15 mg/kg, sc) or the D1R agonist SKF81297 (0.8 mg/kg, sc) until abnormal involuntary movements (AIMs) stabilized. On subsequent test days, rats were treated with vehicle or the 5-HT(1A)R agonist ±8-OH-DPAT (1.0 mg/kg, sc), followed by L-DOPA or SKF81297, or intrastriatal ±8-OH-DPAT (7.5 or 15 mM), followed by L-DOPA. In some cases, the 5-HT(1A)R antagonist WAY100635 was employed to determine receptor-specific effects. In vivo microdialysis was used to collect striatal samples for analysis of extracellular glutamate levels during AIMs assessment. Systemic and striatal ±8-OH-DPAT attenuated L-DOPA-induced dyskinesia and striatal glutamate efflux while WAY100635 reversed ±8-OH-DPAT's effects. Interestingly, systemic ±8-OH-DPAT diminished D1R-mediated AIMs without affecting glutamate. These findings indicate a novel anti-dyskinetic mechanism of action for 5-HT(1A)R agonists with implications for the improved treatment of Parkinson's disease.

Figure 1. L-DOPA- and D1R-mediated priming and post-test AIMs

(A) Rats that received unilateral 6-OHDA lesions of the MFB were primed 3 weeks later with L-DOPA (12 mg/kg + benserazide, 15 mg/kg, sc; Experiments 1 and 3; n = 30) or the D1R agonist SKF81297 (SKF; 0.8 mg/kg,sc; Experiment 2; n = 8) and exhibited summed axial, forelimb, and orolingual (ALO) AIMs ≥ 25 by the last day of priming (3 h total). Following microdialysis test days, rats were given a post-test of L-DOPA or SKF alone in order to ensure AIMs stability over the course of testing. The distribution of axial (white), forelimb (gray), and orolingual (black) AIMs are represented. (B) Timecourse of AIMs for the last day of priming and the post-test of 6-OHDA-lesioned animals treated with L-DOPA or SKF. Differences in AIMs (expressed as medians) were analyzed with non-parametric Kruskal-Wallis tests and Mann-Whitney post hoc comparisons. * p < 0.05 for SKF priming vs L-DOPA priming + p < 0.05 for SKF post-test vs L-DOPA post-test

Figure 2. Effects of 6-OHDA-lesion, L-DOPA priming, and D1R agonist priming on basal extracellular striatal glutamate levels

Rats in the first and second experiments received unilateral 6-OHDA or sham lesions of the MFB and 3 weeks later were primed with L-DOPA (12 mg/kg + benserazide, 15 mg/kg, sc) or the D1R agonist SKF81297 (SKF; 0.8 mg/kg,sc). Following priming, rats underwent a microdialysis procedure including 40 min of baseline sampling prior to any vehicle or drug injection. Striatal glutamate concentrations (M) from these baseline samples were averaged and analyzed with a two-way ANOVA (lesion group × priming treatment). The groups include: 6-OHDA+L-DOPA (n=12), 6-OHDA+SKF (n=9), Sham+L-DOPA (n=11), and Sham+SKF (n=7). * p < 0.05 for 6-OHDA-lesioned groups vs sham-lesioned groups

Figure 3. Effects of 5-HT_1AR stimulation on L-DOPA-related striatal glutamate and AIMs in 6-OHDA- and sham-lesioned rats

Rats in the first experiment received unilateral 6-OHDA or sham lesions of the MFB and 3 weeks later were primed with L-DOPA (12 mg/kg + benserazide, 15 mg/kg, sc) once daily for 7 d. Dyskinetic (n=12) and sham-lesioned rats (n=11) underwent a microdialysis procedure including: 40 min baseline, 120 min vehicle treatment, 120 min drug treatment, and 60 min post-drug treatment sampling (dialysate collected every 20 min). Drug treatment included: Vehicle (VEH; 0.9% NaCl), the full 5-HT_1AR agonist ±8-OH-DPAT (DPAT; 1.0 mg/kg, sc), or combined DPAT + the 5-HT_1AR antagonist WAY100635 (WAY; 0.5 mg/kg, sc), immediately followed by L-DOPA (12 mg/kg, + benserazide, 15 mg/kg, sc). AIMs were observed during this time. Lines depict the means ± SEM of striatal glutamate (percent baseline) (A) and AIMs (C) for VEH+L-DOPA (n=8), DPAT+L-DOPA (n=7), and DPAT+WAY+L-DOPA (n=5) in 6-OHDA-lesioned rats. Results for sham-lesioned rats receiving VEH+L-DOPA (n=7), DPAT+L-DOPA (n=6), and DPAT+WAY+L-DOPA (n=6) are expressed in (D) and (F). Bars depict the means ± SEM concentrations of striatal glutamate (M) during the 120 min of vehicle (VEH+VEH) and 120 min of drug treatments in 6-OHDA- (B) and sham-lesioned (E) rats. Effects over time were determined by two-way mixed design ANOVAs for striatal glutamate (% baseline). Striatal glutamate concentrations (M) were analyzed with one-way ANOVAs. Treatment effects for AIMs (expressed as medians) were analyzed by employing non-parametric Kruskal-Wallis tests. Significant differences between treatments were determined by Mann-Whitney post hoc comparisons for AIMs and planned comparison tests for striatal glutamate (% baseline and M). * p < 0.05 for VEH + L-DOPA vs DPAT + L-DOPA + p < 0.05 for DPAT + L-DOPA vs DPAT + WAY + L-DOPA # p < 0.05 vs VEH + VEH (bar graph)

Figure 4. Effects of 5-HT_1AR stimulation on D1R agonist-related striatal glutamate and AIMs in 6-OHDA- and sham-lesioned rats

Rats in the second experiment received unilateral 6-OHDA or sham lesions of the MFB and 3 weeks later were primed with the D1R agonist SKF81297 (SKF; 0.8 mg/kg, sc) every 2–3 d for a total of 3 d. Dyskinetic (n=9) and sham-lesioned rats (n=7) underwent a microdialysis procedure including: 40 min baseline, 120 min vehicle treatment, 120 min drug treatment, and 60 min post-drug treatment sampling (dialysate collected every 20 min). Drug treatment included: Vehicle (VEH; 0.9% NaCl) or the full 5-HT_1AR agonist ±8-OH-DPAT (DPAT; 1.0 mg/kg, sc), immediately followed by SKF (0.8 mg/kg, sc). AIMs were observed during this time. Lines depict the means ± SEM of striatal glutamate (A) and AIMs (C) for VEH+SKF (n=9) and DPAT+SKF (n=9) in 6-OHDA-lesioned rats. Results for sham-lesioned rats receiving VEH+SKF (n=6) and DPAT+SKF (n=7) are expressed in (D) and (F). Bars depict the means ± SEM concentrations of striatal glutamate (M) during the 120 min of vehicle (VEH+VEH) and 120 min of drug treatments in 6-OHDA- (B) and sham-lesioned (E) rats. Effects over time were determined by two-way mixed design ANOVAs for striatal glutamate (% baseline). Striatal glutamate concentrations (M) were analyzed with oneway ANOVAs. Treatment effects for AIMs (expressed as medians) were analyzed by employing non-parametric Kruskal-Wallis tests. Significant differences between treatments were determined by Mann-Whitney post hoc comparisons for AIMs and planned comparison tests for striatal glutamate (% baseline and M). * p < 0.05 for VEH+SKF vs DPAT+SKF

Figure 5. Effects of striatal 5-HT_1AR stimulation on local glutamate and AIMs in 6-OHDA, L-DOPA-treated rats

Rats in the third experiment received unilateral 6-OHDA of the MFB and 3 weeks later were primed with L-DOPA (12 mg/kg + benserazide, 15 mg/kg, sc) once daily for 7 d. Dyskinetic rats (n=18) underwent a microdialysis procedure including: 40 min baseline, 120 min vehicle treatment, 120 min drug treatment, and 60 min post-drug treatment sampling (dialysate collected every 20 min and expressed as percent baseline). Intrastriatal drug infusion included: Vehicle (VEH; aCSF), the full 5-HT_1AR agonist ±8-OH-DPAT (DPAT; 7.5 or 15 mM), or combined DPAT (15 mM) + WAY100635 (WAY; 4.6 mM), followed 10 min later by systemic treatment injections of L-DOPA (12 mg/kg, + benserazide, 15 mg/kg, sc). AIMs were observed during this time. Lines depict the means ± SEM of striatal glutamate (A) and AIMs (B) for VEH (n=6), DPAT (7.5 mM; n=6), DPAT (15 mM; n=7), and DPAT (15 mM) + WAY (4.6 mM; n=6) groups. Effects over time were determined by a two-way mixed design ANOVA for striatal glutamate. Treatment effects for AIMs (expressed as medians) were analyzed by employing non-parametric Kruskal-Wallis tests. Significant differences between treatments were determined by Mann-Whitney post hoc comparisons for AIMs and planned comparison tests for striatal glutamate. * p < 0.05 for VEH vs DPAT (7.5 mM) ^ p < 0.05 for VEH vs DPAT (15 mM) + p < 0.05 for DPAT (15 mm) vs DPAT (15 mM) + WAY (4.6 mM)