Dopamine receptor agonists regulate levels of the serotonin 5-HT2A receptor and its mRNA in a subpopulation of rat striatal neurons

Abstract

The effects of dopamine receptor agonists on the levels of the striatal serotonin 5-HT2A receptor and its mRNA were investigated in rats lesioned with 6-OHDA as neonates. The mRNA encoding for the 5-HT2A receptor was detected by in situ hybridization histochemistry and the binding of 5-HT2A receptors was revealed with [125I](2,5-dimethoxy-4-iodophenyl)2-aminopropane ([125I]DOI). In adult control unlesioned rats, labeling with the 5-HT2A cRNA probe and with [125I]DOI was concentrated in medial sectors of the striatum. In 6-OHDA-lesioned rats, labeling with the 5-HT2A cRNA probe or with [125I]DOI was increased in the striatum, particularly in its lateral subdivisions. These increases were abolished after chronic systemic administration of the dopamine receptor agonists apomorphine or SKF-38393. The mRNA levels encoding for the 5-HT2A receptor were further measured in individual striatal neurons after double-labeling of sections with a 5-HT2A and a preproenkephalin (PPE) cRNA probe. In control unlesioned rats, 5-HT2A mRNA labeling was distributed in PPE-labeled as well as in PPE-unlabeled striatal neurons. In 6-OHDA-lesioned rats, increased 5-HT2A mRNA labeling was found only in PPE-unlabeled neurons and it was abolished after apomorphine or SKF-38393 administration. These results demonstrate that agonists of dopamine receptors inhibit the expression of 5-HT2A receptors in a subpopulation of presumed striato-nigral neurons. We propose that this regulation plays an important role in the control of motor activity by dopamine and 5-HT in the basal ganglia.

Fig. 1.

Negative images of x-ray films from frontal brain sections processed for in situ hybridization with a³⁵S-labeled 5-HT_2A cRNA probe (exposure time 10 d; A, C, E, G) or incubated with [¹²⁵I]DOI (exposure time 3 d; B, D, F, H). Sections are from adult control sham-operated rats (A, B), adult rats injected with 6-OHDA as neonates (C, D), and from adult rats injected with 6-OHDA as neonates and treated chronically with apomorphine (E, F) or SKF-38393 (G, H). The labeling intensity in each condition was measured by computerized densitometry in the four striatal sectors illustrated in Figure 1B. The surface of analysis shown for the dorsomedial sector was identical for the three other striatal sectors.

Fig. 5.

Bright-field photomicrographs of brain sections processed for in situ hybridization histochemistry with a³⁵S-labeled 5-HT_2A cRNA probe and a digoxigenin-labeled PPE cRNA probe in a ventrolateral striatal sector. Labeling is from an adult control sham-operated rat (A), an adult rat lesioned with 6-OHDA as neonate (B), and an adult rat lesioned with 6-OHDA as neonate and chronically injected with apomorphine (C) or SKF-38393 (D). Neurons labeled with the 5-HT_2AcRNA probe are indicated by the arrows. Note the increased labeling on the PPE-unlabeled neuron of the 6-OHDA-lesioned rat (B). Scale bar, 10 μm.

Fig. 2.

Negative images of x-ray films from frontal brain sections processed for [³H]mazindol (exposure time 3 weeks; A, B) or [³H]citalopram (exposure time 4 weeks; C, D) binding. Sections are from adult control sham-operated rats (A, C) or adult rats injected with 6-OHDA as neonates (B, D).

Fig. 3.

Level of [³H]mazindol or [³H]citalopram binding in the striatum of adult control sham-operated rats (control), adult rats lesioned with 6-OHDA as neonates (lesioned), and adult rats injected with 6-OHDA as neonates and chronically injected with apomorphine, SKF-38393, or a combination of SKF-38393 and SCH-23390. Labeling was measured by computerized densitometry on x-ray film radioautographs. The values represent the average labeling from six rats in each experimental group and are expressed as a percentage of the controls. ANOVAs for [³H]mazindol or [³H]citalopram binding indicated statistical significant differences between experimental groups (F_(4,24) = 4.5, p < 0.0001 andF_(4,24) = 3.1, p < 0.05, respectively). *p < 0.05, **p < 0.005 when compared to controls with the Fisher’s test.

Fig. 4.

Levels of 5-HT_2A mRNA (A, B) or [¹²⁵I]DOI binding (C) in the striatum of adult control sham-operated rats (control), adult rats lesioned with 6-OHDA as neonates (lesioned), and adult rats injected with 6-OHDA as neonates and chronically injected with apomorphine, SKF-38393, or a combination of SKF-38393 and SCH-23390. The values represent the average intensity of labeling measured on x-ray films by computerized densitometry and expressed as a percentage of the controls at frontal levels A = 10 or A = 9.2 according to the stereotaxic atlas of Paxinos and Watson (1986). The data (mean ± SEM) were obtained from six rats in each group. Labeling was measured in four different striatal sectors (DM, dorsomedial; VM, ventromedial; DL, dorsolateral; and VL, ventrolateral). Statistical differences in labeling in each striatal sector were determined after a one-way ANOVA. Pairwise comparisons between different experimental conditions were made according to the Fisher’s test. *, p < 0.01, **,p < 0.005 when compared to the controls; #, p < 0.01, or ##, p < 0.005 when compared to the 6-OHDA-lesioned; and ¶, p < 0.01, or ¶¶, p < 0.005 when compared to the SKF-38393-treated rats.

Fig. 6.

Levels of 5-HT_2A mRNA labeling in single PPE-labeled and PPE-unlabeled neurons in a ventrolateral sector of the striatum. Radioautographic labeling was measured by computerized image analysis (see Materials and Methods for details). The values are means ± SEM of the average number of pixels per neuron and are expressed as a percentage of the controls. Data are from adult control sham-operated rats (control), rats that received 6-OHDA as neonates (lesioned), and rats that received 6-OHDA as neonates and were treated with apomorphine, SKF-38393, or a combination of SKF-38393 and SCH-23390 as adults. A sample of 50 neurons per rat from six rats per experimental condition was analyzed. Pairwise comparisons between experimental groups were made with a Fisher’s test. *, p < 0.01 when compared to the controls; #,p < 0.01 when compared to the lesioned rats; and ¶,p < 0.01 when compared to the SKF-38393-treated rats.

Fig. 7.

Histograms of frequency distributions of 5-HT_2A mRNA labeling in PPE-labeled and PPE-unlabeled neurons of the lateral striatum. Data are from adult control sham-operated rats (Control), rats that received 6-OHDA as neonates (Lesioned), and rats that received 6-OHDA as neonates and were treated with apomorphine, SKF-38393 or a combination of SKF-38393 and SCH-23390 as adults. Quantification of silver grains over individual striatal neurons was performed by computerized image analysis (see Materials and Methods for details). The area covered by silver grains is expressed in number of pixels per neuron. A sample of 50 neurons per rat from six rats in each experimental condition was analyzed.