Postnatal maintenance of the 5-Ht1a-Pet1 autoregulatory loop by serotonin in the raphe nuclei of the brainstem

Abstract

Background: Despite the importance of 5-HT1A as a major target for the action of several anxiolytics/antidepressant drugs, little is known about its regulation in central serotonin (5-hydroxytryptamine, 5-HT) neurons.

Results: We report that expression of 5-HT1A and the transcription factor Pet1 was impaired in the rostral raphe nuclei of mice lacking tryptophan hydroxylase 2 (Tph2) after birth. The downregulation of Pet1 was recapitulated in 5-Ht1a-/- mice. Using an explant culture system, we show that reduction of Pet1 and 5-HT1A was rescued in Tph2-/- brainstem by exogenous 5-HT. In contrast, 5-HT failed to rescue reduced expression of Pet1 in 5-Ht1a-/- brainstem explant culture.

Conclusions: These results suggest a causal relationship between 5-HT1A and Pet1, and reveal a potential mechanism by which 5-HT1A-Pet1 autoregulatory loop is maintained by 5-HT in a spatiotemporal-specific manner during postnatal development. Our results are relevant to understanding the pathophysiology of certain psychiatric and developmental disorders.

Figure 1

Generation and characterization of Tph2^-/- mice. (A) Top: Schematic diagram of the genomic locus for the Tph2 gene. Middle: the gene-targeting construct. Bottom: targeted Tph2 allele. The black box denotes the coding exon. (B) Southern blot analysis of wild-type (Tph2^+/+) and targeted ES clone (Tph2^f/+). Predicted size of the genomic fragments was detected. (C) Growing curve of male and female Tph2^+/+ and Tph2^-/- mice from P1 to 8-week. (D) Anti-5-HT immunoreactivity in the dorsal raphe (left), spinal cord (middle) and intestine (right) of Tph2^+/+ mice (upper row) and Tph2^-/- mice (lower row). (E) Images (left) and quantified data (right) to show in situ hybridization of Tph2, Sert, AADC and VMAT2 in Tph2^+/+ and Tph2^-/- mice. Error bars represent SEM. n = 5. Scale bars, 100 μm.

Figure 2

The expression of Pet1 and Gata3, but not LMX1B was decreased in the dorsal raphe nuclei of Tph2^-/- mice. (A and B) Expression of Pet1(A) and Gata3(B) detected by in situ hybridization in the dorsal and caudal raphe nuclei of Tph2^+/+ mice and Tph2^-/- mice at embryonic day 17.5 (E17.5), postnatal day 0 (P0) and adult stages. (C) Immunohistochemistry images and quantified data to show LMX1B expression in the dorsal and caudal raphe nuclei of adult Tph2^+/+ mice and Tph2^-/- mice. Error bars represent SEM. n = 6. *P < 0.05, versus Tph2^+/+, unpaired t test. Scale bars, 100 μm.

Figure 3

5-HT1A expression was decreased in the dorsal raphe nuclei of Tph2^-/- mice. (A) qRT-PCR analysis of 5Ht1a, 5Ht1b and 5Ht1d in Tph2^+/+ and Tph2^-/- hindbrain. Tph2 mRNA level in the Tph2^-/- hindbrain was used as a negative control. Relative expression was quantified using 18S rRNA as reference gene. (B) Immunostaining of 5-HT1A in the dorsal raphe nucleus, cortex, polymorph layer dentate gyrus (PoDG), inferior colliculus central nucleus (ICc) and superior colliculus (SC) of Tph2^+/+ mice and Tph2^-/- mice. (C) Quantification of 5-HT1A⁺ neurons in B7-8 raphe nucleus. Error bars represent SEM. n = 6. *P < 0.05, versus Tph2^+/+, unpaired t test. Scale bars, 100 μm.

Figure 4

The expression of Pet1 but not Gata3 was decreased in 5Ht1a^-/- mice. (A)In situ hybridization images to show the expression of Pet1, Gata3, Tph2, AADC and VMAT2 and immunostaining of LMX1B in the dorsal raphe nuclei of 5-Ht1a^+/+ mice (upper row) and 5-Ht1a^-/- mice (lower row). (B) Quantification of positive cells for Pet1 and Gata3 in B7-8 raphe nuclei. Error bars represent SEM. n = 6. *P < 0.05, versus 5-Ht1a^+/+, unpaired t test. Scale bar, 100 μm.

Figure 5

The regulation of Pet1 and 5-Ht1a expression by 5-HT in brainstem explants. (A and B) The hindbrain explants of Tph2^-/- mice, 5-Ht1a^-/- mice and their littermates wild-type mice were treated with 5-HT either alone or in combination with WAY-100635, and the expression level of Pet1(A) and 5-Ht1a(B) was measured using qRT-PCR. Relative expression was quantified using 18S rRNA as reference gene. Error bars represent SEM. n = 5. *P < 0.05, versus Saline and ^#P < 0.05, versus 5-HT, one-way ANOVA followed by post hoc analysis. (C) Diagram to show a 5-HT-dependent 5-Ht1a/Pet1 auto regulatory feedback loop.