TrkB receptor controls striatal formation by regulating the number of newborn striatal neurons

Abstract

In the peripheral nervous system, target tissues control the final size of innervating neuronal populations by producing limited amounts of survival-promoting neurotrophic factors during development. However, it remains largely unknown if the same principle works to regulate the size of neuronal populations in the developing brain. Here we show that neurotrophin signaling mediated by the TrkB receptor controls striatal size by promoting the survival of developing medium-sized spiny neurons (MSNs). Selective deletion of the gene for the TrkB receptor in striatal progenitors, using the Dlx5/6-Cre transgene, led to a hindpaw-clasping phenotype and a 50% loss of MSNs without affecting striatal interneurons. This loss resulted mainly from increased apoptosis of newborn MSNs within their birthplace, the lateral ganglionic eminence. Among MSNs, those expressing the dopamine receptor D2 (DRD2) were most affected, as indicated by a drastic loss of these neurons and specific down-regulation of the DRD2 and enkephalin. This specific phenotype of mutant animals is likely due to preferential TrkB expression in DRD2 MSNs. These findings suggest that neurotrophins can control the size of neuronal populations in the brain by promoting the survival of newborn neurons before they migrate to their final destinations.

Fig. 1.

Deletion of the TrkB gene in the striatum leads to a large loss of striatal neurons. (A) Dlx5/6-Cre-mediated recombination pattern in the striatum, as revealed by X-gal staining for β-galactosidase (blue) and DARPP-32 immunohistochemistry (brown) in Rosa26/+;Dlx5/6-Cre mice. (Scale bar, 50 μm.) (B) Western blot shows the complete absence of the full-length TrkB receptor (TrkB-F) and a large reduction in the truncated TrkB receptor (TrkB-T) in the striatum of a mutant mouse. Protein extracts were prepared from striata and cortices of Dlx5/6-Cre-mediated TrkB conditional mutant (M) and fB/fB control (C) mice at 3 wk of age and probed with antibodies against the TrkB extracellular domain and α-tubulin. (C) The TrkB mutant (TrkB^Dlx) at P18 exhibited abnormal hindpaw-clasping phenotype when suspended by its tail. (D) DARPP-32 levels in control (C) and TrkB^Dlx (M) mice, as revealed by Western blot and immunohistochemistry. (Scale bar, 25 μm.) (E) Representative images of Nissl-stained coronal brain sections from control and TrkB^Dlx mice. Stm, striatum; Ctx, cerebral cortex. (Scale bar, 500 μm.) (F) Striatal volumes of TrkB^Dlx and control mice at P21. Note the striatal volume in TrkB^Dlx mice was reduced by 53% compared with control mice (n = 4 mice each). Error bars represent SEM. Student's t test: ***P < 0.001. (G) Counts of striatal neurons in TrkB^Dlx and control mice at P21, obtained from Nissl- or NeuN-stained sections. The striatal neuron counts were reduced by 45% (Nissl) and by 58% (NeuN) compared with control mice (n = 4 mice each). Error bars represent SEM. Student's t test: ***P < 0.001.

Fig. 2.

Differential effects of TrkB deletion on DRD2- and enkephalin-expressing MSNs at P21. (A) Reduced Enk immunoreactivity in the external segment of the globus pallidus (GPe) of TrkB^Dlx mice. (B) Normal SP immunoreactivity in the substantia nigra (SN) of TrkB^Dlx mice. (Scale bar, 250 μm.) (C) Normal expression of DRD1a in the striatum (Stm) of TrkB^Dlx mice. (D) In situ hybridization showing reduced Enk mRNA levels but normal SP mRNA levels in the striatum of TrkB^Dlx mice. (E) Quantification of in situ hybridization signals for Drd2, Enk, Drd1a, and SP mRNAs. Note that levels of mRNAs for DRD2 and Enk but not for DRD1a and SP were significantly reduced in TrkB^Dlx mice (n = 3 mice each). Error bars represent SEM. Student's t test: **P < 0.01; ***P < 0.001. (F) Representative high-magnification images showing fewer EGFP-expressing striatal neurons in fB/fB;Dlx5/6-Cre;D2-EGFP mice (mutant) compared with fB/fB;D2-EGFP mice (control). (Scale bar, 25 μm.) (G) Counts of EGFP-expressing neurons in the striatum. EGFP-expressing striatal neurons were counted in several genotypes of mice to reveal a severe loss of DRD2-expressing MSNs in TrkB mutant mice (n = 4 mice for each genotype). EGFP could be expressed from the Dlx5/6-Cre transgene (Dlx-EGFP) and/or the D2-EGFP transgene. Error bars represent SEM. Student's t test: ***P < 0.001.

Fig. 3.

TrkB is preferentially expressed in DRD2-positive MSNs. (A–C) Colocalization of TrkB with DRD2 in the striatum of adult TrkB^LacZ/+;D2-EGFP mice in which β-galactosidase and EGFP serve as indicators for expression of TrkB and DRD2, respectively. Fluorescent immunohistochemistry with antibodies to β-galactosidase and EGFP shows that the majority of TrkB-expressing neurons also express DRD2 in the adult striatum. (D–F) TrkB expression in striatal MSNs. Antibodies to DARPP-32 and β-galactosidase were used to reveal MSNs and TrkB-expressing cells in the striatum of adult TrkB^LacZ/+ mice. (G–I) High coexpression of TrkB and DRD2 in the young striatum. Nearly all EGFP⁺ cells expressed β-galactosidase in the striatum of TrkB^LacZ/+;D2-EGFP mice at P10. (J–L) Low coexpression of TrkB and DRD1a in the striatum of TrkB^LacZ/+;Drd1a-tdTomato mice at P10. (Scale bar, 50 μm.)

Fig. 4.

Loss of striatal neurons in TrkB^Dlx mice is not due to impaired striatal neurogenesis. (A) Striatal volumes of TrkB^Dlx and control mice at P10 and P0. Striatal volume in TrkB^Dlx mice was reduced by 50% at P10 and 44% at P0 compared with control mice (n = 4 mice each). Error bars represent SEM. Student's t test: **P < 0.01. (B) Striatal neuron counts of TrkB^Dlx and control mice at P10 and P0. Note that the striatal neuron counts in TrkB^Dlx mice was reduced by 40% at P10 and 44% at P0 compared with control mice (n = 4 mice each). Error bars represent SEM. Student's t test: *P < 0.05; **P < 0.01. (C and D) Representative images of BrdU-labeled cells in the VZ and SVZ of the LGE at E16.5. (Scale bar, 25 μm.) (E) Quantification of BrdU-positive cells in the VZ and SVZ of the LGE shows no significant difference between control and TrkB^Dlx embryos at E14.5, E16.5, and E18.5. Error bars represent SEM.

Fig. 5.

Striatal neuronal loss is due to increased apoptosis in the striatal proliferative zone. (A–D) Immunohistochemistry against activated caspase-3 in the VZ/SVZ and striatum at P0. Numerous apoptotic cells were observed in the LGE VZ/SVZ and striatum of TrkB^Dlx mice. Sections were counterstained with Nissl. (E and F) Immunohistochemistry against activated caspase-3 in the LGE VZ/SVZ of embryos at E16.5. (Scale bar, 25 μm.) (G) Density of cells containing activated caspase-3 in the LGE VZ/SVZ and the striatum of control and TrkB^Dlx mice at P0. Error bars represent SEM. Student's t test: ***P < 0.001.