After 3 washes with DPBS, nonspecific protein binding sites were blocked with Blotto (TBS with 4% w/v nonfat dry milk plus 0.1% TX-100) for 1 h at RT and then incubated with primary Abs for 1 h at RT. in L2 and L5a. In addition, L4 of primary somatosensory cortex is strikingly devoid of Kv2.2 immunolabeling. The restricted pattern of Kv2.2 expression persists in Kv2.1-KO mice, suggesting distinct cell- and layer-specific functions for these two highly related Kv2 subunits. Analyses of endogenous Kv2.2 in cortical neurons and recombinant Kv2.2 expressed in heterologous cells reveal that Kv2.2 is largely refractory to stimuli that trigger robust, phosphorylation-dependent changes in Kv2.1 clustering and function. Immunocytochemistry and voltage-clamp recordings from outside-out macropatches reveal distinct cellular expression patterns for Kv2.1 and Kv2.2 in intratelencephalic and pyramidal tract neurons of L5, indicating circuit-specific requirements for these Kv2 paralogs. Together, these results support distinct roles for these two Kv2 channel family members in mammalian cortex. SIGNIFICANCE STATEMENT Neurons within the neocortex are arranged in a laminar architecture and contribute to the input, processing, and/or output of sensory and motor signals in a cell- and layer-specific manner. Neurons of different cortical layers express diverse populations of ion channels and possess distinct intrinsic membrane properties. Here, we show that the Kv2 family members Kv2.1 and Kv2.2 are expressed in distinct cortical layers and pyramidal cell types associated with specific corticostriatal pathways. We find that Kv2.1 and Kv2.2 exhibit distinct responses to acute phosphorylation-dependent regulation in brain neurons and in heterologous cells hybridization (ISH) analyses and single-cell RT-PCR revealed widespread and relatively homogenous expression of Kv2.1 mRNA across cortical layers (Drewe et al., 1992; Hwang et al., 1992; Guan et al., 2007). Immunohistochemical analyses of Kv2.1 expression (Trimmer, 1991; Hwang et al., 1993; Maletic-Savatic et al., 1995; Rhodes et al., 1995; Rhodes et al., Vofopitant (GR 205171) 2004; Mandikian et al., 2014) yielded similar results, although detailed analysis of Kv2.1 cortical expression has not been performed. Functionally, Kv2.1 underlies the bulk of the delayed-rectifier potassium current (and in heterologous cells expressing recombinant Kv2.2. Finally, we show that the expression of Kv2.1 and Kv2.2 is associated with distinct efferent pathways. Together, these results suggest independent roles for these highly related Kv2 channel paralogs in cortical function and plasticity. Materials and Methods Antibodies. See Table 1 for detailed descriptions of Abs used in this study. Table 1. Antibody information of the National Institutes of Health (NIH) and were approved by the University of CaliforniaCDavis (UC-Davis) and the University of Tennessee Health Science Center Institutional Animal Care and Use Committees. Mice and rats were maintained under standard lightCdark cycles and allowed to feed and drink (Misonou et al., 2005). Control mice were anesthetized by pentobarbital (60 mg/kg) without CO2 exposure. Mice were then perfused with 4% formaldehyde (FA) for immunohistochemistry (see below). We NFKBIA have previously shown that CO2 inhalation and global decapitation ischemia exhibit a similar extent of Kv2.1 modulation (Misonou et al., 2005). For preparation of brain sections, rats and mice were deeply anesthetized with 60 mg/kg sodium pentobarbital and transcardially perfused with 5 ml PBS (150 mm NaCl, 10 mm Na-phosphate buffer, pH 7.4) containing 10 U/ml heparin, followed Vofopitant (GR 205171) by 30 ml ice-cold 4% FA (freshly prepared from PFA) in 0.1 m sodium phosphate buffer, pH 7.4 (0.1 m PB). The brains were removed and cryoprotected for 24 h in 10% sucrose and then for 24C48 Vofopitant (GR 205171) h in 30% sucrose in 0.1 m PB. Perfusion-fixed and cryoprotected ferret brains were gifts from the laboratory of our late colleague, Dr. Barbara Chapman. Fresh-frozen macaque samples were a gift from the laboratory of our late colleague, Dr. Edward G. Jones. Fresh-frozen human brain samples (49.5-year-old Caucasian male, 5 h postmortem interval) were obtained from the Eunice Kennedy Shriver National Institute of Child Health and Human Development Brain and Tissue Bank for Developmental Disorders (NICHD contract HHSN275200900011C, reference NO1-HD-9-0011). Samples from the visual cortex of human and macaque were thawed in 4% FA, freshly prepared from PFA, in 0.1 m PB, pH 7.4, fixed for 45 min at 4C, and cryoprotected for 24 h in 10% sucrose and then for 48 h in 30% sucrose. After cryoprotection, all samples were.