Endogenous calretinin interneurons make up of

Endogenous calretinin+ interneurons make up 0.5% of striatal neurons in the rodent but are more prevalent in humans (Rymar et al., 2004; Wu and Parent, 2000). These neurons are diverse in morphology with limited electrophysiological characterization (Tepper et al., 2010). Electrophysiological analyses show that the induced neurons are heterogeneous, although the majority is hyperpolarized with lower input resistance and higher AP amplitude and frequency. This heterogeneity may reflect functional diversity and/or the progressive nature of neuronal maturation and integration into the local circuits. Interestingly, a minority of the recorded neurons exhibit pacemaker-like activity and fire spontaneous APs at resting membrane potential. This activity is prevalent in striatal cholinergic neurons, subthalamic nucleus neurons, midbrain dopaminergic neurons, cerebellar Purkinje and Golgi neurons, and low-threshold spike interneurons of the striatum (Beatty et al., 2012; Reynolds and Wickens, 2004; Sharott et al., 2012). The pace-making activity of these neurons can synchronize neuronal networks for higher-order brain functions (Ramirez et al., 2004).
Experimental Procedures
Author Contributions
Acknowledgments We thank members of the C.-L.Z. laboratory for discussions and reagents, Dr. F. Guillemot for sharing the Ascl1 mice, and Dr. M. Klymkowsky for SOX3 antibody. C-.L.Z. is a W. W. Caruth, Jr. Scholar in Biomedical Research. This work was supported by the Welch Foundation Award (I-1724), the Ellison Medical Foundation Award (AG-NS-0753-11), Texas Institute for Brain Injury and Repair, the Decherd Foundation, and NIH grants (NS070981 and NS088095 to C.-L.Z. and NS032817 to J.E.J.).
Introduction The complex structure of the mammalian cerebral cortex is derived from neuroepithelial (NE) cells in the neural tube (McConnell, 1995). NE cells give birth to multiple progenitor populations (Götz and Huttner, 2005; McConnell, 1995). There are two germinal zones in the embryonic neocortex: the ventricular zone (VZ) and the subventricular zone (SVZ) (Gal et al., 2006). Radial glial (RG) cells give rise to self-renewing cells and produce intermediate progenitor (IP) cells via Lomustine division. IP cells subsequently divide into two neurons via symmetrical division (Götz and Huttner, 2005; McConnell, 1995; Rakic, 1995). During the process of progenitor cell transformation into mature neurons, the precise control of the timing of self-renewal, differentiation, neuronal migration, and neuronal maturation of neural progenitor cells (NPCs) is required (Xu et al., 2014). Therefore, it is not surprising that mistakes in this process of early cortical development lead to serious consequences, such as autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD). Metabotropic glutamate receptor 7 (GRM7) is defined as an ASD- (Yang and Pan, 2013) and ADHD-related gene (Elia et al., 2012) and is exclusively expressed in the CNS (Bradley et al., 1996). Metabotropic glutamate receptors are potential targets for neuropsychiatric disorders (Dev, 2004) that modulate neurotransmitter release and neuronal excitability (Schlett, 2006). Metabotropic glutamate receptors are subdivided into groups I (GRM1 and GRM5), II (GRM2 and GRM3), and III (GRM4, GRM6, GRM7, and GRM8) on the basis of homology, intracellular messengers, and ligand selectivity (Schlett, 2006). Characteristic of all metabotropic glutamate receptors, the GRM7 protein is localized to the neuronal presynaptic membrane, and its protein sequence is highly conserved (Bradley et al., 1996). These findings suggest that GRM7 may play an important and irreplaceable role in the nervous system. However, its role in the process of cortical development is unclear. During neurogenesis, cyclic AMP response element-binding protein (CREB) is involved in multiple aspects of neuronal development and plasticity, including cell survival, proliferation, and differentiation (Mantamadiotis et al., 2012). CREB is expressed throughout neurogenesis (Giachino et al., 2005), and a previous study has shown that neural proliferation defects result from the alteration of CREB activity during early development (Dworkin et al., 2007). Yes-associated protein (YAP) modulates organ size by regulating cell apoptosis and proliferation (Cai et al., 2010; Lian et al., 2010). YAP is expressed in mitotic neuronal progenitors, and it is downregulated during neuronal differentiation (Zhang et al., 2012). The phosphorylation of YAP at Ser127 results in a loss of function and the subsequent repression of downstream target genes, leading to premature neuronal differentiation (Cao et al., 2008). In the absence of inhibitory phosphorylation, YAP promotes cell proliferation and suppresses cell differentiation (Zhang et al., 2012). During neurogenesis, CYCLIND1 plays an important role in neural progenitor proliferation; when CYCLIND1 is constitutively activated, the proliferation of NPCs is increased (Das et al., 2010). To investigate the function of GRM7 in early cortical development, we downregulated its expression in neuronal progenitor cells of the cerebral ventricle of embryos via in utero electroporation (IUE). We determined that Grm7 knockdown increases the proliferation of PAX6-positive RG cells, decreases the amplification of TBR2-positive IP cells, and results in a reduction in the number of progenitor cells that differentiate into neurons. Furthermore, morphological maturation was seriously affected by the silencing of Grm7. We also demonstrated that Creb or Yap knockdown ameliorates the Grm7 knockdown phenotype in vivo. Overall, our findings suggest that GRM7 regulates the phosphorylation of CREB and the expression of YAP in neuronal progenitor cells, affecting the expression of CyclinD1, which ultimately controls neuronal differentiation and maturation during cortical development.