Pcdhαc2 is required for axonal tiling and assembly of serotonergic circuitries in mice

Abstract

Serotonergic neurons project their axons pervasively throughout the brain and innervate various target fields in a space-filling manner, leading to tiled arrangements of their axon terminals to allow optimal allocation of serotonin among target neurons. Here we show that conditional deletion of the mouse protocadherin α (Pcdhα) gene cluster in serotonergic neurons disrupts local axonal tiling and global assembly of serotonergic circuitries and results in depression-like behaviors. Genetic dissection and expression profiling revealed that this role is specifically mediated by Pcdhαc2, which is the only Pcdhα isoform expressed in serotonergic neurons. We conclude that, in contrast to neurite self-avoidance, which requires single-cell identity mediated by Pcdh diversity, a single cell-type identity mediated by the common C-type Pcdh isoform is required for axonal tiling and assembly of serotonergic circuitries.

Fig. 1. Characterization of the Pcdhα gene cluster–deletion mice

(A) The mouse Pcdhα, Pcdhβ, and Pcdhγ gene clusters contain 14, 22, and 22 variable exons, respectively, each encoding the extracellular domain (ECD), transmembrane domain, and variable intracellular domain (ICD) of a Pcdh protein. In the Pcdhα and Pcdhγ gene clusters, these variable exons are spliced to three cluster-specific constant exons that encode the common ICD of the corresponding protein isoforms. All 14 Pcdhα variable exons are deleted from the Pcdhα gene cluster (boxed with red dashed line). (B) Depression-related behaviors in Pcdhα-deficient mice, including increased immobility time in TST, FST, and CFC tests. Average immobility times for minutes 3 to 6 in the TST and FST are shown in bar graphs. *P < 0.05; **P < 0.01; ***P < 0.001. N = 16 to 25 per genotype group. (C) Schematic drawing of the serotonergic pathways in the brain. Serotonergic cell bodies and axonal projections are depicted in red. (D) Pcdhα proteins are enriched in serotonergic neurons (TPH2+), as shown with the Pcdhα^mCherry/+ reporter mice. DR, dorsal raphe. (E) Serotonergic wiring defects in Pcdhα-deficient mice. Shown are serotonergic axon terminals (SERT+) in target fields, as indicated. Examples of serotonergic fiber clumps [crossovers on a single optical section (Z = 0.5 µm)] and patterns of redistribution are indicated with red asterisks and arrows, respectively. Scale bars, 100 µm. SI, substantia innominata; act, anterior commissure, temporal limb; CA, cornu ammonis; DG, dentate gyrus; SR, stratum radiatum; SLM, stratum lacunosum moleculare; MO, molecular layer.

Fig. 2. Both serotonergic-wiring and behavioral defects result from Pcdhα loss of function in serotonergic neurons

(A and B) Serotonergic-wiring patterns in Pcdhα conditional knockouts. Whereas serotonergic-innervation patterns remain unchanged in forebrain-specific knockouts (Pcdhα^f/f; Camk2a::Cre), the serotonergic-wiring phenotype is recapitulated in serotonergic-specific knockouts (Pcdhα^f/f; Slc6a4::Cre). Scale bars, 100 µm. (C) 3D visualization (scale bar, 100 µm) and reconstruction (scale bar, 5 µm) of Brainbow AAV–labeled Pcdhα-deficient serotonergic axon terminals in target fields. Fiber clumps are indicated with white asterisks. mTFP, TagBFP, mCherry, and EYFP are reporter genes encoding green, blue, red and yellow fluorescent proteins, respectively. (D) Depression-related behaviors in Pcdhα conditional knockouts. Whereas no significant differences were observed in forebrain-specific knockouts, the behavioral phenotype was recapitulated in serotonergic-specific knockouts. *P < 0.05; **P < 0.01; N.S., not significant. N = 17 to 22 per genotype group.

Fig. 3. Serotonergic wiring is specifically mediated by Pcdhα C-type isoforms

(A and B) Pcdhβ and Pcdhγ gene clusters are not required for serotonergic wiring in the brain. No alterations in serotonergic-innervation patterns were observed in Pcdhβ-cluster knockouts (A) or serotonergic-specific Pcdhγ conditional knockouts (B). (C and D) Genetic dissection of Pcdhα gene cluster. Whereas mice lacking all 12 Pcdhα alternative isoforms display normal serotonergic-innervation patterns (C), the serotonergic-wiring phenotype is recapitulated in Pcdhα C-type–isoform knockouts (D). Scale bars, 100 µm.

Fig. 4. Pcdhαc2 is the predominantly expressed Pcdh isoform in serotonergic neurons

(A) Bulk expression levels of variable exons of the corresponding clustered Pcdh isoforms in serotonergic neurons, as shown with TRAP-Seq. RPKM, reads per kilobase of transcript per million mapped reads. (B) Single-cell expression profiles of clustered Pcdh isoforms in various subtypes of serotonergic neurons. Shown here are neurons with high levels of marker-gene expression (Slc6a4 and Tph2, CPM > 5000). CPM, counts per million mapped reads.