Multimodal Single-Cell Analysis Reveals Physiological Maturation in the Developing Human Neocortex

•Multimodal analysis differentiates cells beyond transcriptomic classification•Single-cell analysis links stimulus-induced calcium elevations to transcriptomes•Cell-type-specific responses to neurotransmitters are associated with maturation•Serotonergic signaling in human radial glia promotes radial fiber formation In the developing human neocortex, progenitor cells generate diverse cell types prenatally. Progenitor cells and newborn neurons respond to signaling cues, including neurotransmitters. While single-cell RNA sequencing has revealed cellular diversity, physiological heterogeneity has yet to be mapped onto these developing and diverse cell types. By combining measurements of intracellular Ca2+ elevations in response to neurotransmitter receptor agonists and RNA sequencing of the same single cells, we show that Ca2+ responses are cell-type-specific and change dynamically with lineage progression. Physiological response properties predict molecular cell identity and additionally reveal diversity not captured by single-cell transcriptomics. We find that the serotonin receptor HTR2A selectively activates radial glia cells in the developing human, but not mouse, neocortex, and inhibiting HTR2A receptors in human radial glia disrupts the radial glial scaffold. We show highly specific neurotransmitter signaling during neurogenesis in the developing human neocortex and highlight evolutionarily divergent mechanisms of physiological signaling. In the developing human neocortex, progenitor cells generate diverse cell types prenatally. Progenitor cells and newborn neurons respond to signaling cues, including neurotransmitters. While single-cell RNA sequencing has revealed cellular diversity, physiological heterogeneity has yet to be mapped onto these developing and diverse cell types. By combining measurements of intracellular Ca2+ elevations in response to neurotransmitter receptor agonists and RNA sequencing of the same single cells, we show that Ca2+ responses are cell-type-specific and change dynamically with lineage progression. Physiological response properties predict molecular cell identity and additionally reveal diversity not captured by single-cell transcriptomics. We find that the serotonin receptor HTR2A selectively activates radial glia cells in the developing human, but not mouse, neocortex, and inhibiting HTR2A receptors in human radial glia disrupts the radial glial scaffold. We show highly specific neurotransmitter signaling during neurogenesis in the developing human neocortex and highlight evolutionarily divergent mechanisms of physiological signaling. The neocortex has expanded in mammalian evolution through an increase in the number of progenitor cells and the time period over which they produce neurons and glia (Azevedo et al., 2009Azevedo F.A.C. Carvalho L.R.B. Grinberg L.T. Farfel J.M. Ferretti R.E.L. Leite R.E.P. Jacob Filho W. Lent R. Herculano-Houzel S. Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain.J. Comp. Neurol. 2009; 513: 532-541Crossref PubMed Scopus (724) Google Scholar). Human radial glia cells, the neural stem cells of the developing cortex, have been subdivided into several subtypes characterized by distinct morphologies, dynamic behavior, and transcriptomic profiles (Lui et al., 2011Lui J.H. Hansen D.V. Kriegstein A.R. Development and evolution of the human neocortex.Cell. 2011; 146: 18-36Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar, Pollen et al., 2015Pollen A.A. Nowakowski T.J. Chen J. Retallack H. Sandoval-Espinosa C. Nicholas C.R. Shuga J. Liu S.J. Oldham M.C. Diaz A. et al.Molecular identity of human outer radial glia during cortical development.Cell. 2015; 163: 55-67Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, Thomsen et al., 2016Thomsen E.R. Mich J.K. Yao Z. Hodge R.D. Doyle A.M. Jang S. Shehata S.I. Nelson A.M. Shapovalova N.V. Levi B.P. Ramanathan S. Fixed single-cell transcriptomic characterization of human radial glial diversity.Nat. Methods. 2016; 13: 87-93Crossref PubMed Scopus (74) Google Scholar). Ventricular radial glia (vRG) are embedded in an adhesion belt along the ventricle and maintain ventricular contact. Outer radial glia (oRG) (Fietz et al., 2010Fietz S.A. Kelava I. Vogt J. Wilsch-Bräuninger M. Stenzel D. Fish J.L. Corbeil D. Riehn A. Distler W. Nitsch R. Huttner W.B. OSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling.Nat. Neurosci. 2010; 13: 690-699Crossref PubMed Scopus (376) Google Scholar, Hansen et al., 2010Hansen D.V. Lui J.H. Parker P.R.L. Kriegstein A.R. Neurogenic radial glia in the outer subventricular zone of human neocortex.Nature. 2010; 464: 554-561Crossref PubMed Scopus (587) Google Scholar) in the outer subventricular zone (OSVZ) arise from vRGs through a process resembling epithelial-mesenchymal transition. oRGs are very rare in mice (Shitamukai et al., 2011Shitamukai A. Konno D. Matsuzaki F. Oblique radial glial divisions in the developing mouse neocortex induce self-renewing progenitors outside the germinal zone that resemble primate outer subventricular zone progenitors.J. Neurosci. 2011; 31: 3683-3695Crossref PubMed Scopus (225) Google Scholar, Wang et al., 2011Wang X. Tsai J.-W. LaMonica B. Kriegstein A.R. A new subtype of progenitor cell in the mouse embryonic neocortex.Nat. Neurosci. 2011; 14: 555-561Crossref PubMed Scopus (236) Google Scholar) and are thought to have significantly contributed to the evolutionary expansion of the neocortex in primates and carnivores (Lui et al., 2011Lui J.H. Hansen D.V. Kriegstein A.R. Development and evolution of the human neocortex.Cell. 2011; 146: 18-36Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar). Intermediate progenitor cells (IPCs) are multipolar transit amplifying cells generated by radial glia that directly produce most of the excitatory neurons in the neocortex (Hansen et al., 2010Hansen D.V. Lui J.H. Parker P.R.L. Kriegstein A.R. Neurogenic radial glia in the outer subventricular zone of human neocortex.Nature. 2010; 464: 554-561Crossref PubMed Scopus (587) Google Scholar, Kowalczyk et al., 2009Kowalczyk T. Pontious A. Englund C. Daza R. Bedogni F. Hodge R.D. Attardo A. Bell C. Huttner W.B. Hevner R.F. Intermediate neuronal progenitors (basal progenitors) produce pyramidal–projection neurons for all layers of cerebral cortex.Cereb. Cortex. 2009; 19: 2439-2450Crossref PubMed Scopus (0) Google Scholar, Noctor et al., 2004Noctor S.C. Martínez-Cerdeño V. Ivic L. Kriegstein A.R. Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases.Nat. Neurosci. 2004; 7: 136-144Crossref PubMed Scopus (1327) Google Scholar). Newborn excitatory neurons migrate along radial glial fibers from the germinal zones (GZ) to the cortical plate (CP), eventually forming six layers (Rakic, 2000Rakic P. Radial unit hypothesis of neocortical expansion.Novartis Found. Symp. 2000; 228 (discussion 42–52): 30-42Crossref PubMed Google Scholar), while inhibitory interneurons originate predominantly from the ganglionic eminences and migrate tangentially into the neocortex (Anderson et al., 2001Anderson S.A. Marín O. Horn C. Jennings K. Rubenstein J.L. Distinct cortical migrations from the medial and lateral ganglionic eminences.Development. 2001; 128: 353-363Crossref PubMed Google Scholar, Hansen et al., 2013Hansen D.V. Lui J.H. Flandin P. Yoshikawa K. Rubenstein J.L. Alvarez-Buylla A. Kriegstein A.R. Non-epithelial stem cells and cortical interneuron production in the human ganglionic eminences.Nat. Neurosci. 2013; 16: 1576-1587Crossref PubMed Scopus (121) Google Scholar). Neurotransmitters can induce Ca2+ transients and influence progenitor cell proliferation, neuronal migration, and differentiation in rodents (Le Magueresse and Monyer, 2013Le Magueresse C. Monyer H. GABAergic interneurons shape the functional maturation of the cortex.Neuron. 2013; 77: 388-405Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, LoTurco et al., 1995LoTurco J.J. Owens D.F. Heath M.J.S. Davis M.B.E. Kriegstein A.R. GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis.Neuron. 1995; 15: 1287-1298Abstract Full Text PDF PubMed Scopus (824) Google Scholar, Weissman et al., 2004Weissman T.A. Riquelme P.A. Ivic L. Flint A.C. Kriegstein A.R. Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex.Neuron. 2004; 43: 647-661Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Progenitor cells, maturing excitatory neurons and interneurons, endothelial cells, and the cerebrospinal fluid (CSF) are all potential sources of neurotransmitters (Bonnin et al., 2011Bonnin A. Goeden N. Chen K. Wilson M.L. King J. Shih J.C. Blakely R.D. Deneris E.S. Levitt P. A transient placental source of serotonin for the fetal forebrain.Nature. 2011; 472: 347-350Crossref PubMed Scopus (249) Google Scholar, Le Magueresse and Monyer, 2013Le Magueresse C. Monyer H. GABAergic interneurons shape the functional maturation of the cortex.Neuron. 2013; 77: 388-405Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, Lehtinen et al., 2011Lehtinen M.K. Zappaterra M.W. Chen X. Yang Y.J. Hill A.D. Lun M. Maynard T. Gonzalez D. Kim S. Ye P. et al.The cerebrospinal fluid provides a proliferative niche for neural progenitor cells.Neuron. 2011; 69: 893-905Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, Li et al., 2018Li S. Kumar T P. Joshee S. Kirschstein T. Subburaju S. Khalili J.S. Kloepper J. Du C. Elkhal A. Szabó G. et al.Endothelial cell-derived GABA signaling modulates neuronal migration and postnatal behavior.Cell Res. 2018; 28: 221-248Crossref PubMed Scopus (4) Google Scholar, Manent et al., 2005Manent J.-B. Demarque M. Jorquera I. Pellegrino C. Ben-Ari Y. Aniksztejn L. Represa A. A noncanonical release of GABA and glutamate modulates neuronal migration.J. Neurosci. 2005; 25: 4755-4765Crossref PubMed Scopus (159) Google Scholar, Reillo et al., 2017Reillo I. de Juan Romero C. Cárdenas A. Clascá F. Martínez-Martinez M.Á. Borrell V. A complex code of extrinsic influences on cortical progenitor cells of higher mammals.Cereb. Cortex. 2017; 27: 4586-4606Crossref PubMed Scopus (3) Google Scholar, Weissman et al., 2004Weissman T.A. Riquelme P.A. Ivic L. Flint A.C. Kriegstein A.R. Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex.Neuron. 2004; 43: 647-661Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Additionally, axons from extracortical regions such as the diencephalon and brain stem innervate the cortex and affect cortical development (Bonetti and Surace, 2010Bonetti C. Surace E.M. Mouse embryonic retina delivers information controlling cortical neurogenesis.PLoS ONE. 2010; 5: e15211Crossref PubMed Scopus (12) Google Scholar, Chen and Kriegstein, 2015Chen J. Kriegstein A.R. A GABAergic projection from the zona incerta to cortex promotes cortical neuron development.Science. 2015; 350: 554-558Crossref PubMed Scopus (60) Google Scholar, Duque et al., 2016Duque A. Krsnik Z. Kostović I. Rakic P. Secondary expansion of the transient subplate zone in the developing cerebrum of human and nonhuman primates.Proc. Natl. Acad. Sci. USA. 2016; 113: 9892-9897Crossref PubMed Scopus (15) Google Scholar, Reillo et al., 2017Reillo I. de Juan Romero C. Cárdenas A. Clascá F. Martínez-Martinez M.Á. Borrell V. A complex code of extrinsic influences on cortical progenitor cells of higher mammals.Cereb. Cortex. 2017; 27: 4586-4606Crossref PubMed Scopus (3) Google Scholar). Protracted development in humans may suggest a greater role of neurotransmitter signaling in regulating cellular processes. Differences in activity-dependent gene expression as well as differences in gene expression in serotonergic and cholinergic systems between different mammalian species indicate that neurotransmitter signaling has likely impacted cortical evolution (Ataman et al., 2016Ataman B. Boulting G.L. Harmin D.A. Yang M.G. Baker-Salisbury M. Yap E.-L. Malik A.N. Mei K. Rubin A.A. Spiegel I. et al.Evolution of Osteocrin as an activity-regulated factor in the primate brain.Nature. 2016; 539: 242-247Crossref PubMed Scopus (27) Google Scholar, Sousa et al., 2017Sousa A.M.M. Zhu Y. Raghanti M.A. Kitchen R.R. Onorati M. Tebbenkamp A.T.N. Stutz B. Meyer K.A. Li M. Kawasawa Y.I. et al.Molecular and cellular reorganization of neural circuits in the human lineage.Science. 2017; 358: 1027-1032Crossref PubMed Scopus (6) Google Scholar). The diverse cell types in the developing neocortex likely respond differently to neurotransmitter signals. In the developing human neocortex, single-cell RNA sequencing (scRNA-seq) has revealed molecular signatures of distinct cell types (Nowakowski et al., 2017Nowakowski T.J. Bhaduri A. Pollen A.A. Alvarado B. Mostajo-Radji M.A. Di Lullo E. Haeussler M. Sandoval-Espinosa C. Liu S.J. Velmeshev D. et al.Spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex.Science. 2017; 358: 1318-1323Crossref PubMed Scopus (17) Google Scholar, Pollen et al., 2015Pollen A.A. Nowakowski T.J. Chen J. Retallack H. Sandoval-Espinosa C. Nicholas C.R. Shuga J. Liu S.J. Oldham M.C. Diaz A. et al.Molecular identity of human outer radial glia during cortical development.Cell. 2015; 163: 55-67Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, Thomsen et al., 2016Thomsen E.R. Mich J.K. Yao Z. Hodge R.D. Doyle A.M. Jang S. Shehata S.I. Nelson A.M. Shapovalova N.V. Levi B.P. Ramanathan S. Fixed single-cell transcriptomic characterization of human radial glial diversity.Nat. Methods. 2016; 13: 87-93Crossref PubMed Scopus (74) Google Scholar). Neuronal identity, however, is further determined by properties beyond gene expression, including morphology, connectivity, and physiology. Therefore, large-scale analyses of neuronal cell diversity will require multimodal characterization of cells that overlay physiological properties and transcriptomic signatures at the single-cell level. This has proven challenging due to technical difficulties (Regev et al., 2017Regev A. Teichmann S.A. Lander E.S. Amit I. Benoist C. Yosef N. Birney E. Bodenmiller B. Campbell P. Carninci P. et al.The human cell atlas.Elife. 2017; 6: e27041Crossref PubMed Scopus (157) Google Scholar, Zeng and Sanes, 2017Zeng H. Sanes J.R. Neuronal cell-type classification: challenges, opportunities and the path forward.Nat. Rev. Neurosci. 2017; 18: 530-546Crossref PubMed Scopus (77) Google Scholar). However, combining scRNA-seq with patch-clamp recording has enabled the integration of intrinsic physiological and molecular properties in relatively few cells (Cadwell et al., 2016Cadwell C.R. Palasantza A. Jiang X. Berens P. Deng Q. Yilmaz M. Reimer J. Shen S. Bethge M. Tolias K.F. et al.Electrophysiological, transcriptomic and morphologic profiling of single neurons using Patch-seq.Nat. Biotechnol. 2016; 34: 199-203Crossref PubMed Scopus (94) Google Scholar, Chen et al., 2016Chen X. Zhang K. Zhou L. Gao X. Wang J. Yao Y. He F. Luo Y. Yu Y. Li S. et al.Coupled electrophysiological recording and single cell transcriptome analyses revealed molecular mechanisms underlying neuronal maturation.Protein Cell. 2016; 7: 175-186Crossref PubMed Scopus (1) Google Scholar, Fuzik et al., 2016Fuzik J. Zeisel A. Máté Z. Calvigioni D. Yanagawa Y. Szabó G. Linnarsson S. Harkany T. Integration of electrophysiological recordings with single-cell RNA-seq data identifies neuronal subtypes.Nat. Biotechnol. 2016; 34: 175-183Crossref PubMed Scopus (82) Google Scholar). We asked whether molecularly distinct cell types in the developing human neocortex also display unique physiological properties. To link neurotransmitter signaling to molecular profiles, we developed a multimodal single-cell approach that allows agonist dosing, response monitoring, and subsequent transcriptomic analysis in the same single cells. Using intracellular calcium elevations as a proxy for physiological responsiveness to a series of neurotransmitter receptor agonists, we found that not only do physiological response properties correlate with molecular identity, but they also capture additional aspects of diversity. Interestingly, inhibition of a serotonergic receptor expressed in human, but not mouse, radial glia disrupts the radial glial scaffold. Together, these findings highlight the cell-type specificity and functional importance of early neurotransmitter-mediated signaling events in the developing human neocortex and provide an avenue for analyzing early active signaling networks. To identify cell-type-specific neurotransmitter receptor systems in the developing human neocortex, we first analyzed the expression of neurotransmitter receptor genes in a scRNA-seq dataset (Table S1) (Nowakowski et al., 2017Nowakowski T.J. Bhaduri A. Pollen A.A. Alvarado B. Mostajo-Radji M.A. Di Lullo E. Haeussler M. Sandoval-Espinosa C. Liu S.J. Velmeshev D. et al.Spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex.Science. 2017; 358: 1318-1323Crossref PubMed Scopus (17) Google Scholar). While not all receptors are likely to be detectable with scRNA-seq, we did find receptor gene transcripts enriched in specific cell populations of the excitatory lineage using pseudoage analysis (Figure 1A). Two N-methyl-D-aspartic acid (NMDA) receptor subunits had opposing expression patterns: GRIN2A was highly expressed in progenitors, while GRIN2B expression was only detected in neurons (Figure 1B), consistent with findings from rodent neural stem cells (Muth-Köhne et al., 2010aMuth-Köhne E. Terhag J. Pahl S. Werner M. Joshi I. Hollmann M. Functional excitatory GABAA receptors precede ionotropic glutamate receptors in radial glia-like neural stem cells.Mol. Cell. Neurosci. 2010; 43: 209-221Crossref PubMed Scopus (12) Google Scholar). The transition from GRIN2A to GRIN2B expression yields channels with faster kinetics and less sensitivity and is important for developmental regulation of synaptic plasticity and the maturation of neuronal circuits (Flint et al., 1997Flint A.C. Maisch U.S. Weishaupt J.H. Kriegstein A.R. Monyer H. NR2A subunit expression shortens NMDA receptor synaptic currents in developing neocortex.J. Neurosci. 1997; 17: 2469-2476Crossref PubMed Google Scholar, Muth-Köhne et al., 2010bMuth-Köhne E. Pachernegg S. Karus M. Faissner A. Hollmann M. Expression of NMDA receptors and Ca2+-impermeable AMPA receptors requires neuronal differentiation and allows discrimination between two different types of neural stem cells.Cell. Physiol. Biochem. 2010; 26: 935-946Crossref PubMed Scopus (12) Google Scholar, Paoletti et al., 2013Paoletti P. Bellone C. Zhou Q. NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease.Nat. Rev. Neurosci. 2013; 14: 383-400Crossref PubMed Scopus (830) Google Scholar). Similarly, we detected a developmental switch in gamma-amino butyric acid (GABA) A receptor subunit expression. GABRB1 and GABRG1 subunits were expressed in progenitor cells, a subunit combination yielding higher affinity, non-desensitizing GABA responses (Ma and Barker, 1995Ma W. Barker J.L. Complementary expressions of transcripts encoding GAD67 and GABAA receptor α4, βl, and γl subunits in the proliferative zone of the embryonic rat central nervous system.J. Neurosci. 1995; 15: 2547-2560Crossref PubMed Google Scholar, Owens et al., 1999Owens D.F. Liu X. Kriegstein A.R. Porcher C. Hatchett C. Longbottom R.E. Mcainch K. Talvinder S. Moss S.J. Thomson A.M. et al.Changing properties of GABA A receptor-mediated signaling during early neocortical development.J. Neurophysiol. 1999; 82: 570-583Crossref PubMed Scopus (137) Google Scholar), while characteristic synaptic subunits GABRB3 and GABRG2 were expressed in neurons (Figure 1C) (Luscher et al., 2011Luscher B. Fuchs T. Kilpatrick C.L. GABAA receptor trafficking-mediated plasticity of inhibitory synapses.Neuron. 2011; 70: 385-409Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). The purinergic P2RY1 receptor was highly expressed in progenitor cells, including radial glia and IPCs, and decreased sharply in neurons (Figure 1D), consistent with reports in rodents (Liu et al., 2008Liu X. Hashimoto-Torii K. Torii M. Haydar T.F. Rakic P. The role of ATP signaling in the migration of intermediate neuronal progenitors to the neocortical subventricular zone.Proc. Natl. Acad. Sci. USA. 2008; 105: 11802-11807Crossref PubMed Scopus (0) Google Scholar, Weissman et al., 2004Weissman T.A. Riquelme P.A. Ivic L. Flint A.C. Kriegstein A.R. Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex.Neuron. 2004; 43: 647-661Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GRIA1-3 expression increased as cells differentiated (Figure 1E), in line with increased chromatin accessibility in the promoter regions of GRIA1-4 in the CP compared to the GZ (de la Torre-Ubieta et al., 2018de la Torre-Ubieta L. Stein J.L. Won H. Opland C.K. Liang D. Lu D. Geschwind D.H. The dynamic landscape of open chromatin during human cortical neurogenesis.Cell. 2018; 172: 289-304Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). We find that neurotransmitter receptor subunits of the same receptor type (e.g., different NMDA receptor subunits) have different and sometimes opposing expression trajectories in the human neocortex that correlate with the development of distinct physiological properties. We found that HTR2A, a receptor not previously reported in progenitor cells (Vitalis and Verney, 2017Vitalis T. Verney C. Sculpting cerebral cortex with serotonin in rodent and primate.in: Serotonin - A Chemical Messenger Between All Types of Living Cells. InTech, 2017Crossref Google Scholar), was highly expressed in first and second trimester human radial glia (Figure 1F). The expression of HTR2A correlated with genes important for progenitor cell proliferation including CTNNA1 and SMARCC2 (Table S1) (Tuoc et al., 2013Tuoc T.C. Boretius S. Sansom S.N. Pitulescu M.E. Frahm J. Livesey F.J. Stoykova A. Chromatin regulation by BAF170 controls cerebral cortical size and thickness.Dev. Cell. 2013; 25: 256-269Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, Lien et al., 2006Lien W.H. Klezovitch O. Fernandez T.E. Delrow J. Vasioukhin V. alphaE-catenin controls cerebral cortical size by regulating the hedgehog signaling pathway.Science. 2006; 311: 1609-1612Crossref PubMed Scopus (187) Google Scholar). Because vRG and oRG cells reside in distinct progenitor regions, we aimed to identify expression of HTR2A in specific laminae by single-molecule fluorescent in situ hybridization (smFISH). Starting at postconceptional week 8 (PCW8), HTR2A was strongly expressed in the ventricular and subventricular zones (Figures 1G, S1A, and S1C). Additionally, HTR2A was expressed in the CP at midgestation (Figures 1G and S1C). By performing immunohistochemistry after smFISH, we found that HTR2A colocalized with the vRG marker CRYAB (Figure 1H) and was enriched in cells positive for the oRG marker HOPX (Figure 1I and S1D). HTR2A also colocalized with the mitotic marker Ki67 (Figure 1J) but only rarely with the IPC marker PPP1R17 (Figure 1J). The Htr2a receptor is not expressed during neurogenesis in developing mouse neocortex (Figure S1E), indicating the potential evolutionary importance of this receptor in human radial glia. In contrast, P2RY1, which is expressed in progenitors in rodents (Liu et al., 2008Liu X. Hashimoto-Torii K. Torii M. Haydar T.F. Rakic P. The role of ATP signaling in the migration of intermediate neuronal progenitors to the neocortical subventricular zone.Proc. Natl. Acad. Sci. USA. 2008; 105: 11802-11807Crossref PubMed Scopus (0) Google Scholar, Weissman et al., 2004Weissman T.A. Riquelme P.A. Ivic L. Flint A.C. Kriegstein A.R. Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex.Neuron. 2004; 43: 647-661Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar), was also expressed in the human GZ (Figures S1A–S1C), but not enriched in oRGs (Figure S1D). These findings indicate that, in addition to displaying unique molecular signatures (Pollen et al., 2015Pollen A.A. Nowakowski T.J. Chen J. Retallack H. Sandoval-Espinosa C. Nicholas C.R. Shuga J. Liu S.J. Oldham M.C. Diaz A. et al.Molecular identity of human outer radial glia during cortical development.Cell. 2015; 163: 55-67Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, Thomsen et al., 2016Thomsen E.R. Mich J.K. Yao Z. Hodge R.D. Doyle A.M. Jang S. Shehata S.I. Nelson A.M. Shapovalova N.V. Levi B.P. Ramanathan S. Fixed single-cell transcriptomic characterization of human radial glial diversity.Nat. Methods. 2016; 13: 87-93Crossref PubMed Scopus (74) Google Scholar), human oRG cells may also be responsive to serotonergic stimulation. We next tested whether receptor mRNAs expressed by neural progenitor cells and immature neurons are translated into functional receptors. Based on transcriptomic (Figures 1 and S1) and physiological studies (Flint et al., 1999Flint A.C. Dammerman R.S. Kriegstein A.R. Endogenous activation of metabotropic glutamate receptors in neocortical development causes neuronal calcium oscillations.Proc. Natl. Acad. Sci. USA. 1999; 96: 12144-12149Crossref PubMed Scopus (0) Google Scholar, Haydar et al., 2000Haydar T.F. Wang F. Schwartz M.L. Rakic P. Differential modulation of proliferation in the neocortical ventricular and subventricular zones.J. Neurosci. 2000; 20: 5764-5774Crossref PubMed Google Scholar, Liu et al., 2008Liu X. Hashimoto-Torii K. Torii M. Haydar T.F. Rakic P. The role of ATP signaling in the migration of intermediate neuronal progenitors to the neocortical subventricular zone.Proc. Natl. Acad. Sci. USA. 2008; 105: 11802-11807Crossref PubMed Scopus (0) Google Scholar, LoTurco et al., 1995LoTurco J.J. Owens D.F. Heath M.J.S. Davis M.B.E. Kriegstein A.R. GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis.Neuron. 1995; 15: 1287-1298Abstract Full Text PDF PubMed Scopus (824) Google Scholar, Ma et al., 2000Ma W. Maric D. Li B.S. Hu Q. Andreadis J.D. Grant G.M. Liu Q.Y. Shaffer K.M. Chang Y.H. Zhang L. et al.Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation.Eur. J. Neurosci. 2000; 12: 1227-1240Crossref PubMed Scopus (136) Google Scholar, Weissman et al., 2004Weissman T.A. Riquelme P.A. Ivic L. Flint A.C. Kriegstein A.R. Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex.Neuron. 2004; 43: 647-661Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar), we compiled a panel of neurotransmitter receptor agonists active during brain development that included two specific glutamate receptor agonists, NMDA and AMPA, as well as GABA, acetylcholine (ACh), and purinergic P2RY1 receptor agonist methylthioadenosine diphosphate (MeSADP). Additionally, we included TCB-2, a specific agonist for HTR2A, whose activation induces membrane currents and intracellular Ca2+ elevations in pyramidal neurons and astrocytes (Aghajanian and Marek, 1999Aghajanian G.K. Marek G.J. Serotonin, via 5-HT2A receptors, increases EPSCs in layer V pyramidal cells of prefrontal cortex by an asynchronous mode of glutamate release.Brain Res. 1999; 825: 161-171Crossref PubMed Scopus (225) Google Scholar, Hagberg et al., 1998Hagberg G.B. Blomstrand F. Nilsson M. Tamir H. Hansson E. Stimulation of 5-HT2A receptors on astrocytes in primary culture opens voltage-independent Ca2+ channels.Neurochem. Int. 1998; 32: 153-162Crossref PubMed Scopus (0) Google Scholar). We used patch-clamp recordings in second trimester acute cortical slices to measure currents induced by these neurotransmitter receptor agonists. Cell types were identified based on their anatomical location, electrophysiological properties, morphology, and marker gene expression (Figures 2A, 2B, and S2A–S2C). We found a diversity of cellular responses to agonist application. Consistent with Mg2+ block of the NMDA receptor at hyperpolarized membrane potentials, NMDA only induced currents at positive membrane potentials in neurons (Figure 2C). However, NMDA induced currents at resting membrane potentials in radial glia (Figure 2C), consistent with Mg2+-insensitive NMDA currents recorded in immature neurons, such as subplate (SP) neurons in the developing rodent brain (Hanganu et al., 2002Hanganu I.L. Kilb W. Luhmann H.J. Functional synaptic projections onto subplate neurons in neonatal rat somatosensory cortex.J. Neurosci. 2002; 22: 7165-7176Crossref PubMed Google Scholar), as well as glial cells (Káradóttir et al., 2005Káradóttir R. Cavelier P. Bergersen L.H. Attwell D. NMDA receptors are expressed in oligodendrocytes and activated in ischaemia.Nature. 2005; 438: 1162-1166Crossref PubMed Scopus (494) Google Scholar, Lalo et al., 2006Lalo U. Pankratov Y. Kirchhoff F. North R.A. Verkhratsky A. NMDA receptors mediate neuron-to-glia signaling in mouse cortical astrocytes.J. Neurosci. 2006; 26: 2673-2683Crossref PubMed Scopus (240) Google Scholar). AMPA and GABA induced currents in all cell types analyzed (Figure 2C). TCB-2 induced currents in vRGs and oRGs, while MeSADP induced hyperpolarization only in vRGs (Figure 2C). Receptor specificity was confirmed by antagonists that selectively blocked agonist-induced currents (Figures S2C–S2I). GABA-induced currents in vRGs were larger than in oRGs, which we hypothesized to be due to gap junctio