Chronic L-dopa decreases serotonin neurons in a subregion of the dorsal raphe nucleus

Abstract

L-Dopa (l-3,4-dihydroxyphenylalanine) is the precursor to dopamine and has become the mainstay therapeutic treatment for Parkinson's disease. Chronic L-dopa is administered to recover motor function in Parkinson's disease patients. However, drug efficacy decreases over time, and debilitating side effects occur, such as dyskinesia and mood disturbances. The therapeutic effect and some of the side effects of L-dopa have been credited to its effect on serotonin (5-HT) neurons. Given these findings, it was hypothesized that chronic L-dopa treatment decreases 5-HT neurons in the dorsal raphe nucleus (DRN) and the content of 5-HT in forebrain regions in a manner that is mediated by oxidative stress. Rats were treated chronically with l-dopa (6 mg/kg; twice daily) for 10 days. Results indicated that the number of 5-HT neurons was significantly decreased in the DRN after l-dopa treatment compared with vehicle. This effect was more pronounced in the caudal-extent of the dorsal DRN, a subregion found to have a significantly higher increase in the 3,4-dihydroxyphenylacetic acid/dopamine ratio in response to acute L-dopa treatment. Furthermore, pretreatment with ascorbic acid (400 mg/kg) or deprenyl (2 mg/kg) prevented the l-dopa-induced decreases in 5-HT neurons. In addition, 5-HT content was decreased significantly in the DRN and prefrontal cortex by l-dopa treatment, effects that were prevented by ascorbic acid pretreatment. Taken together, these data illustrate that chronic L-dopa causes a 5-HT neuron loss and the depletion of 5-HT content in a subregion of the DRN as well as in the frontal cortex through an oxidative-stress mechanism.

Fig. 1.

Effects of l-dopa on TPH⁺/NeuN⁺ cell counts in the DRN. Rats received 10 days of l-dopa or vehicle treatment. (A) Chronic l-dopa treatment significantly decreased TPH⁺/NeuN⁺ cell bodies (*P < 0.05 compared with vehicle, Student’s t test). (B) Representative DRN images (original magnification: 20×) illustrating TPH⁺/NeuN⁺ colabeling (green = NeuN⁺, red = TPH⁺, yellow = TPH⁺/NeuN⁺ colabel) (n = 9–11 per group).

Fig. 2.

Subregional effects of l-dopa on TPH⁺ cell counts in the DRN. (A) Chronic l-dopa treatment had no effect on TPH⁺/NeuN⁺ cell bodies compared with vehicle in the rostral-extent of the dorsal subregion. (B) l-Dopa treatment significantly decreased TPH⁺/NeuN⁺ cell bodies in the caudal-extent of the dorsal subregion (*P < 0.05 compared with vehicle, Student’s t test). (C and D) l-Dopa treatment had no effect on TPH⁺/NeuN cell bodies compared with vehicle in the either rostral or caudal-extent of the ventral subregion (n = 9–11 per group).

Fig. 3.

Effects of ascorbic acid and deprenyl on l-dopa induced decreases in TPH⁺ cell counts. TPH⁺/NeuN⁺ cell bodies were counted within the caudal-extent of the dorsal DRN subregion. (A) Both ascorbic acid (400 mg/kg) and deprenyl (2 mg/kg) prevented the effects of l-dopa at decreasing TPH⁺/NeuN⁺ cell bodies (*P < 0.05 compared with vehicle, two-way ANOVA and Tukey’s post-hoc test). (B) Representative DRN images (original magnification: 20×) illustrating TPH⁺/NeuN⁺ colabeling (green = NeuN⁺, red = TPH⁺, yellow = TPH⁺/NeuN⁺ colabel) (n = 4–6 per group).

Fig. 4.

Acute l-dopa and dopamine tissue content in the DRN. Dopamine, DOPAC, and HVA tissue content in the DRN was measured 45 minutes after l-dopa injection. (A and B) Acute l-dopa significantly elevated dopamine and its metabolites in both the dorsal and ventral DRN compared with vehicle. (C) The magnitude of dopamine increase after acute l-dopa did not differ between dorsal and ventral DRN. (D) The DOPAC/dopamine ratio was significantly greater in the dorsal compared with ventral DRN after acute l-dopa. (*P < 0.05 compared with vehicle controls; ^#P < 0.05 compared with ventral DRN, Student’s t test) (n = 6 per group).

Fig. 5.

Chronic l-dopa and 5-HT tissue content. Rats received 10 days of l-dopa or vehicle treatment. Chronic l-dopa significantly reduced 5-HT tissue content in the dorsal DRN and the PFC (*P < 0.05 compared with vehicle, Student’s t test). l-Dopa treatment did not affect the ventral DRN, striatum, or hippocampus (n = 8–12 per group).

Fig. 6.

Effects of ascorbic acid on l-dopa induced decreases in 5-HT tissue content. (A) Ascorbic acid prevented chronic l-dopa–induced decreases in 5-HT tissue content in the dorsal DRN. (B) Ascorbic acid pretreatment also prevented the 5-HT tissue content decreases in the PFC (*P < 0.05 compared with vehicle, two-way ANOVA and Tukey’s post-hoc test) (n = 9–12 per group).