Slitrk5 deficiency impairs corticostriatal circuitry and leads to obsessive-compulsive-like behaviors in mice

Abstract

Obsessive-compulsive disorder (OCD) is a common psychiatric disorder defined by the presence of obsessive thoughts and repetitive compulsive actions, and it often encompasses anxiety and depressive symptoms. Recently, the corticostriatal circuitry has been implicated in the pathogenesis of OCD. However, the etiology, pathophysiology and molecular basis of OCD remain unknown. Several studies indicate that the pathogenesis of OCD has a genetic component. Here we demonstrate that loss of a neuron-specific transmembrane protein, SLIT and NTRK-like protein-5 (Slitrk5), leads to OCD-like behaviors in mice, which manifests as excessive self-grooming and increased anxiety-like behaviors, and is alleviated by the selective serotonin reuptake inhibitor fluoxetine. Slitrk5(-/-) mice show selective overactivation of the orbitofrontal cortex, abnormalities in striatal anatomy and cell morphology and alterations in glutamate receptor composition, which contribute to deficient corticostriatal neurotransmission. Thus, our studies identify Slitrk5 as an essential molecule at corticostriatal synapses and provide a new mouse model of OCD-like behaviors.

Figure 1

Targeted inactivation of Slitrk5 in mice and its expression pattern in the mouse brain. (a) Genomic structure and the design of the Slitrk5-knockout, lacZ–knock-in mouse. The entire open reading frame (ORF) is localized to exon 2 (Ex2); exon 1 (Ex1) is noncoding. The Slitrk5-encoding region was replaced with lacZ downstream of the signal sequence cleavage site. WT, wild-type; KO, knockout. (b) X-gal staining of mouse brain tissue, showing ubiquitous expression of lacZ in the gray matter of the various parts of the brain, including cortex and striatum. Cx, cortex; St, striatum; Hp, hippocampus; cc, corpus callosum; Th, thalamus; Cbl, cerebellum. The higher magnification image shows the distribution of lacZ-expressing cells in the striatum of the Slitrk5-knockout, lacZ–knock-in mouse. (c) Immunostaining of cortex and striatum with antibodies to β-galactosidase (anti–β-gal) and NeuN (anti-NeuN), indicating that the majority of neurons express Slitrk5.

Figure 2

Facial lesions, OCD-like behavior and its alleviation with fluoxetine treatment in Slitrk5-knockout mice. (a) Phenotypic characteristic of Slitrk5^−/− mice: excessive grooming leads to severe facial lesions. (b) Time spent grooming in Slitrk5^−/− mice (n = 9) compared to their wild-type littermates (n = 8) before and after treatment with fluoxetine. Error bars depict the s.e.m. (c) Anxiety-related behavior of Slitrk5^−/− and WT mice in the open-field test. Percentage of time spent in the center and entries into the center of the open field are shown. All open-field results are presented as means ± s.e.m. determined from analysis of 20 mice per genotype.

Figure 3

Metabolic changes in the cortex and anatomical defects in the striatum of Slitrk5^−/− mice. (a) Expression of FosB in orbitofrontal cortex by immunostaining for FosB (red) and with DAPI (blue). The top images show the distribution of FosB expression in the various layers of orbitofrontal cortex. The bottom images show a higher magnification of layer II of FosB immunoreactivity in nuclei. (b) Quantification of FosB expression in all layers of the orbitofrontal cortex. (c) Cavalieri estimation of striatal volume in Slitrk5^−/− and WT mice. (d) Examples of Golgi staining and Neurolucida reconstruction of striatal medium spiny neurons in WT and Slitrk5^−/− mice. (e) Sholl analysis of striatal medium spiny neurons in WT and Slitrk5^−/− mice. All results are presented as means ± s.e.m.; 40 neurons per genotype. (f) Fractal dimension analysis of striatal medium spiny neurons in Slitrk5^−/− and WT mice. All results are presented as means ± s.e.m.; 40 neurons per genotype.

Figure 4

Deficiency in corticostriatal transmission in Slitrk5^−/− mice is mediated by changes in glutamate receptor composition. (a) Immunostaining of primary striatal rat neurons (infected with Flag-Slitrk5 lentivirus and transfected with PSD95 fused to mCherry (PSD95-cherry)) in culture with cortical neurons (isolated from transgenic mice that ubiquitously express green fluorescent protein) with Flag-specific antibody (anti-Flag). The arrow points to a magnified area (bottom images) that represents the synapses between cortical and striatal neurons. (b) Western blot analysis of NMDA and AMPA receptor subunits in the striatum of 5-month-old Slitrk5^−/− and WT mice. The protein amounts are adjusted to the expression of actin. (c) Population spike amplitude in Slitrk5^−/− mice (n = 11, from four mice) and matched WT mice (n = 9, from four mice). The population spike amplitude is significantly lower in Slitrk5^−/− mice, P < 0.01, repeated-measures analysis of variance. The inset shows examples of corticostriatal population spike amplitudes in Slitrk5^−/− mice and matched WT mice. (d) Average paired-pulse ratios of the population spike in Slitrk5^−/− mice (n = 17, from five mice) and matched WT mice (n = 17, from five mice). There is no significant difference in the paired-pulse ratio between Slitrk5^−/− mice and wild-type mice.