Traditionally, neuroscientists have defined the identity of neurons by the cells' location, morphology, connectivity and excitability. However, the direct relationship between these parameters and the molecular phenotypes has remained largely unexplored. Here, we present a method for obtaining full transcriptome data from single neocortical pyramidal cells and interneurons after whole-cell patch-clamp recordings in mouse brain slices. In our approach, termed Patch-seq, a patch-clamp stimulus protocol is followed by the aspiration of the entire somatic compartment into the recording pipette, reverse transcription of RNA including addition of unique molecular identifiers, cDNA amplification, Illumina library preparation and sequencing. We show that Patch-seq reveals a close link between electrophysiological characteristics, responses to acute chemical challenges and RNA expression of neurotransmitter receptors and channels. Moreover, it distinguishes neuronal subpopulations that correspond to both well-established and, to our knowledge, hitherto undescribed neuronal subtypes. Our findings demonstrate the ability of Patch-seq to precisely map neuronal subtypes and predict their network contributions in the brain.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Gene Expression Omnibus
Fishell, G. & Hanashima, C. Pyramidal neurons grow up and change their mind. Neuron 57, 333–338 (2008).
Rakic, P. Evolution of the neocortex: a perspective from developmental biology. Nat. Rev. Neurosci. 10, 724–735 (2009).
Klausberger, T. & Somogyi, P. Neuronal diversity and temporal dynamics: the unity of hippocampal circuit operations. Science 321, 53–57 (2008).
Tricoire, L. et al. A blueprint for the spatiotemporal origins of mouse hippocampal interneuron diversity. J. Neurosci. 31, 10948–10970 (2011).
Freund, T.F. & Buzsáki, G. Interneurons of the hippocampus. Hippocampus 6, 347–470 (1996).
Ascoli, G.A. et al. Petilla Interneuron Nomenclature Group. Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex. Nat. Rev. Neurosci. 9, 557–568 (2008).
Cauli, B. et al. Molecular and physiological diversity of cortical nonpyramidal cells. J. Neurosci. 17, 3894–3906 (1997).
Cauli, B. et al. Classification of fusiform neocortical interneurons based on unsupervised clustering. Proc. Natl. Acad. Sci. USA 97, 6144–6149 (2000).
Gupta, A., Wang, Y. & Markram, H. Organizing principles for a diversity of GABAergic interneurons and synapses in the neocortex. Science 287, 273–278 (2000).
Markram, H. The blue brain project. Nat. Rev. Neurosci. 7, 153–160 (2006).
Zeisel, A. et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 1138–1142 (2015).
Romanov, R.A. et al. A secretagogin locus of the mammalian hypothalamus controls stress hormone release. EMBO J. 34, 36–54 (2015).
Usoskin, D. et al. Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing. Nat. Neurosci. 18, 145–153 (2015).
Okaty, B.W., Sugino, K. & Nelson, S.B. Cell type-specific transcriptomics in the brain. J. Neurosci. 31, 6939–6943 (2011).
Okaty, B.W., Sugino, K. & Nelson, S.B. A quantitative comparison of cell-type-specific microarray gene expression profiling methods in the mouse brain. PLoS One 6, e16493 (2011).
Subkhankulova, T., Yano, K., Robinson, H.P. & Livesey, F.J. Grouping and classifying electrophysiologically-defined classes of neocortical neurons by single cell, whole-genome expression profiling. Front. Mol. Neurosci. 3, 10 (2010).
Citri, A., Pang, Z.P., Südhof, T.C., Wernig, M. & Malenka, R.C. Comprehensive qPCR profiling of gene expression in single neuronal cells. Nat. Protoc. 7, 118–127 (2012).
Veys, K., Labro, A.J., De Schutter, E. & Snyders, D.J. Quantitative single-cell ion-channel gene expression profiling through an improved qRT-PCR technique combined with whole cell patch clamp. J. Neurosci. Methods 209, 227–234 (2012).
Qiu, S. et al. Single-neuron RNA-Seq: technical feasibility and reproducibility. Front. Genet. 3, 124 (2012).
Freund, T.F. Interneuron Diversity series: Rhythm and mood in perisomatic inhibition. Trends Neurosci. 26, 489–495 (2003).
Hashimoto, T. et al. Alterations in GABA-related transcriptome in the dorsolateral prefrontal cortex of subjects with schizophrenia. Mol. Psychiatry 13, 147–161 (2008).
Schmidt, M.J. et al. Modulation of behavioral networks by selective interneuronal inactivation. Mol. Psychiatry 19, 580–587 (2014).
Tamamaki, N. et al. Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse. J. Comp. Neurol. 467, 60–79 (2003).
Máté, Z. et al. Spatiotemporal expression pattern of DsRedT3/CCK gene construct during postnatal development of myenteric plexus in transgenic mice. Cell Tissue Res. 352, 199–206 (2013).
Tainaka, K. et al. Whole-body imaging with single-cell resolution by tissue decolorization. Cell 159, 911–924 (2014).
Susaki, E.A. et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157, 726–739 (2014).
Tomer, R., Ye, L., Hsueh, B. & Deisseroth, K. Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat. Protoc. 9, 1682–1697 (2014).
Kawaguchi, Y. & Kubota, Y. Neurochemical features and synaptic connections of large physiologically-identified GABAergic cells in the rat frontal cortex. Neuroscience 85, 677–701 (1998).
Islam, S. et al. Highly multiplexed and strand-specific single-cell RNA 5′ end sequencing. Nat. Protoc. 7, 813–828 (2012).
Islam, S. et al. Quantitative single-cell RNA-seq with unique molecular identifiers. Nat. Methods 11, 163–166 (2014).
Chaudhry, F.A. et al. The vesicular GABA transporter, VGAT, localizes to synaptic vesicles in sets of glycinergic as well as GABAergic neurons. J. Neurosci. 18, 9733–9750 (1998).
Fremeau, R.T. Jr. et al. The expression of vesicular glutamate transporters defines two classes of excitatory synapse. Neuron 31, 247–260 (2001).
Kaneko, T., Fujiyama, F. & Hioki, H. Immunohistochemical localization of candidates for vesicular glutamate transporters in the rat brain. J. Comp. Neurol. 444, 39–62 (2002).
Kirischuk, S., Parpura, V. & Verkhratsky, A. Sodium dynamics: another key to astroglial excitability? Trends Neurosci. 35, 497–506 (2012).
Arganda, S., Guantes, R. & de Polavieja, G.G. Sodium pumps adapt spike bursting to stimulus statistics. Nat. Neurosci. 10, 1467–1473 (2007).
Mindell, J.A. & Maduke, M. ClC chloride channels. Genome Biol. 2, S3003 (2001).
Baranauskas, G., Tkatch, T., Nagata, K., Yeh, J.Z. & Surmeier, D.J. Kv3.4 subunits enhance the repolarizing efficiency of Kv3.1 channels in fast-spiking neurons. Nat. Neurosci. 6, 258–266 (2003).
Angulo, M.C., Lambolez, B., Audinat, E., Hestrin, S. & Rossier, J. Subunit composition, kinetic, and permeation properties of AMPA receptors in single neocortical nonpyramidal cells. J. Neurosci. 17, 6685–6696 (1997).
Tsou, K., Brown, S., Sañudo-Peña, M.C., Mackie, K. & Walker, J.M. Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system. Neuroscience 83, 393–411 (1998).
Katona, I. et al. Presynaptically located CB1 cannabinoid receptors regulate GABA release from axon terminals of specific hippocampal interneurons. J. Neurosci. 19, 4544–4558 (1999).
Muñoz-Manchado, A.B. et al. Novel Striatal GABAergic Interneuron Populations Labeled in the 5HT3aEGFP Mouse. Cereb. Cortex doi:10.1093/cercor/bhu179 (21 August 2014).
Varga, V. et al. Fast synaptic subcortical control of hippocampal circuits. Science 326, 449–453 (2009).
Férézou, I. et al. 5-HT3 receptors mediate serotonergic fast synaptic excitation of neocortical vasoactive intestinal peptide/cholecystokinin interneurons. J. Neurosci. 22, 7389–7397 (2002).
Caiati, M.D. & Cherubini, E. Fluoxetine impairs GABAergic signaling in hippocampal slices from neonatal rats. Front. Cell. Neurosci. 7, 63 (2013).
Tamás, G., Buhl, E.H., Lörincz, A. & Somogyi, P. Proximally targeted GABAergic synapses and gap junctions synchronize cortical interneurons. Nat. Neurosci. 3, 366–371 (2000).
Rakic, P. A century of progress in corticoneurogenesis: from silver impregnation to genetic engineering. Cereb. Cortex 16 (suppl. 1), i3–i17 (2006).
Morozov, Y.M. & Freund, T.F. Postnatal development and migration of cholecystokinin-immunoreactive interneurons in rat hippocampus. Neuroscience 120, 923–939 (2003).
Zivraj, K.H. et al. Subcellular profiling reveals distinct and developmentally regulated repertoire of growth cone mRNAs. J. Neurosci. 30, 15464–15478 (2010).
Holt, C.E. & Schuman, E.M. The central dogma decentralized: new perspectives on RNA function and local translation in neurons. Neuron 80, 648–657 (2013).
Schwanhäusser, B. et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011).
We thank A. Juréus for DNA sequencing, and the CLICK Imaging Facility at Karolinska Institutet for making the Imaris software package available for neuronal reconstructions, T. Klausberger and E. Borók for discussions and assistance with Neurolucida reconstructions. This work was supported by the European Research Council (BRAINCELL 261063, to S.L.), the Swedish Research Council (to S.L. and T.H.); Human Frontier Science Program (to A.Z.), the European Commission 7th Framework Program (PAINCAGE, to T.H.), Hjärnfonden (to T.H.) and the NovoNordisk Foundation (to T.H.).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Distribution and molecular heterogeneity of dual-labeled interneurons in the postnatal mouse brain.
(a) Overview of CCKBAC/dsRed:GAD67gfp/+ mouse somatosensory cortex on postnatal day 20, revealing the distribution of CCK+ (magenta) and/or GAD67+ (green) cells in the different cortical layers (L; labeled from L1-L6a). Light pink/white color depicts co-localization. Overview image of the mouse forebrain (left) was reconstructed from tiled confocal photomicrographs using a Zeiss LSM780 laser-scanning microscope. Open rectangle indicates the location of the inset (right). Scale bars = 500 μm (left) and 200 μm (right). (b-d) Representative current-clamp recordings of CCK+/GAD67− pyramidal cells (Exc L2/3 (b), Exc L4 (c) and Exc L5 (d)). At the left of each panel, AP responses (top) to square current pulses (bottom) are shown. Phase-plane plots of the APs rising upon 2x rheobase current injection (top right) and rheobasic APs (bottom right) are depicted for each cell type. In phase-plane plots, the first AP is red, while subsequent APs shift from warm to cool blue color. For the rheobasic AP, the y-axis between -20 mV and +30 mV was omitted to emphasize AHP and ADP characteristics (scale bars = 200 pA (vertical), 25 ms (horizontal)). (e) Cell-type-specific expression of a voltage gated K+ channel interacting protein (Kcnip1), a GTPase-activating protein (Chn1), a protein kinase C substrate (Nrgn), a Ca2+ channel subunit (Cacna2d3), a Na+ channel subunit (Scn3a), Purkinje cell protein 4 (Pcp4), a G protein-signaling regulator (Rgs12), serotonin receptor subtype 3a (Htr3a), reelin (Reln), a superficial layer-specific marker, calbindin D28k (Calb1), a Ca2+-binding protein, vasoactive intestinal polypeptide (Vip) and neuropeptide Y (Npy) in sub-classified I-type CCK+ interneurons (magenta) and Exc-type pyramidal cells (green).
Step-wise improvements to the sampling, collection, ejection and analysis protocols are shown. Overall, 145 neurons were used to optimize recording and processing conditions, while ~120 neurons were processed to obtain reliable RNA-seq data. Axis labels on Bioanalyzer (Agilent) plots are: [FU], fluorescence unit; (bp) base pair. Magenta-colored “x” labels steps that had been omitted due to poorer outcomes.
Supplementary Figure 3 Linear regression of genes implicated in resting membrane potential and sub-threshold electrical events.
(a) Positive linear regression between Atp1a3 (subunit of the Na+/K+-ATPase) and Vrest. (b) Likewise, positive and close relationship between quantitative expression of Clcn3, a voltage-gated Cl− channel subunit, and Vrest. Each data point represents the two-dimensional population mean for the parameters indicated. Standard deviations were not plotted to retain maximum visual clarity.
1Mindell, J.A. & Maduke, M. ClC chloride channels. Genome Biol. 2, REVIEWS 3003 (2001).
Supplementary Figure 4 Correlation of action potential (AP) parameters and mRNA expression for synaptic proteins, receptor subunits.
(a) Heat map of correlation coefficients (scaled, color-coded and filtered to <−0.4 or >0.4 and from >5 cells) of mRNA counts and characteristic electrophysiological properties of dual-labeled interneurons. Open rectangles denote significant examples in our predictive matrix, which are shown in (d,e). (b) Color matrix marks the relationship of individual parameters and particular time-locked phases of a single action AP or AP waveforms (in c). (d,e) Correlation between electrophysiology parameters and gene expression of Syt7 or Gria1. Neuronal subclass identity is shown by color-coding. (f-i) Correlation (f-h) and anti-correlation (i) of quantitative Kcnc1 expression and a subset of biophysical membrane parameters during the 1st AP.
2Zeisel, A. et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 1138-1142 (2015).
About this article
Cite this article
Fuzik, J., Zeisel, A., Máté, Z. et al. Integration of electrophysiological recordings with single-cell RNA-seq data identifies neuronal subtypes. Nat Biotechnol 34, 175–183 (2016). https://doi.org/10.1038/nbt.3443
Transcriptional and morphological profiling of parvalbumin interneuron subpopulations in the mouse hippocampus
Nature Communications (2021)
Pten is a key intrinsic factor regulating raphe 5-HT neuronal plasticity and depressive behaviors in mice
Translational Psychiatry (2021)
Expression of serotonin 1A and 2A receptors in molecular- and projection-defined neurons of the mouse insular cortex
Molecular Brain (2020)
Human iPSC-derived mature microglia retain their identity and functionally integrate in the chimeric mouse brain
Nature Communications (2020)