Single-cell RNAseq reveals cell adhesion molecule profiles in electrophysiologically defined neurons Csaba Földya,b,1, Spyros Darmanisc, Jason Aotoa,d, Robert C. Malenkae, Stephen R. Quakec,f, and Thomas C. Südhofa,f,1 aDepartment of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305; bBrain Research Institute, University of Zürich, 8057 Zurich, Switzerland; cDepartment of Bioengineering, Stanford University, Stanford, CA 94305; dDepartment of Pharmacology, University of Colorado Denver, Aurora, CO 80045; eNancy Pritzker Laboratory, Stanford University, Stanford, CA 94305; and fHoward Hughes Medical Institute, Stanford University, Stanford, CA 94305 Contributed by Thomas C. Südhof, July 10, 2016 (sent for review May 21, 2016; reviewed by Thomas Biederer, Tamas F. Freund, and Li-Huei Tsai) In brain, signaling mediated by cell adhesion molecules defines the neurexin (Nrxn1 and Nrxn3; presynaptic cell adhesion mole- identity and functional properties of synapses. The specificity of cules) isoforms were expressed cell type-specifically, with re- presynaptic and postsynaptic interactions that is presumably medi- markable consistency in respective cell types (9). We also found ated by cell adhesion molecules suggests that there exists a logic that that genetic deletion of neuroligin-3 (Nlgn3) (postsynaptic cell could explain neuronal connectivity at the molecular level. Despite its adhesion molecule) in PYR cells disabled tonic, cannabinoid importance, however, the nature of such logic is poorly understood, type 1 receptor-mediated, endocannabinoid signaling in RS CCK and even basic parameters, such as the number, identity, and single- synapses, but had no detectable phenotype in FS PV synapses cell expression profiles of candidate synaptic cell adhesion molecules, (10). Thus, although no systematic assessment of cell adhesion are not known. Here, we devised a comprehensive list of genes molecules in GABAergic interneurons is available, previous studies established that cell adhesion molecules play a central involved in cell adhesion, and used single-cell RNA sequencing role in controlling their properties. (RNAseq) to analyze their expression in electrophysiologically de- Similar to their inputs, outputs of CA1-PYR cells display fined interneurons and projection neurons. We compared the cell functional dichotomy: they primarily project to the subiculum, type-specific expression of these genes with that of genes involved forming synapses on two, electrophysiologically different prin- in transmembrane ion conductances (i.e., channels), exocytosis, and cipal cell types: regular-spiking pyramidal (RS-PYR) and burst- rho/rac signaling, which regulates the actin cytoskeleton. Using spiking pyramidal (BS-PYR) cells. Analysis of these neurons is these data, we identified two independent, developmentally regu- particularly difficult because, although RS-PYR and BS-PYR lated networks of interacting genes encoding molecules involved in cells exhibit distinct electrophysiological signatures as well as cell adhesion, exocytosis, and signal transduction. Our approach pro- dramatically different forms of long-term plasticity, no molecular vides a framework for a presumed cell adhesion and signaling code markers are available to distinguish these neurons (11, 12). In in neurons, enables correlating electrophysiological with molecular examining synapse-specific mechanisms in these cells, we have properties of neurons, and suggests avenues toward understanding shown that different forms of long-term plasticity were de- synaptic specificity. termined presynaptically by expression of specific neurexin iso- forms in CA1-PYR cells (13–15). Together, these molecular and synapse | cell adhesion | single cell | RNAseq physiological analyses revealed specific control of synaptic properties (such as forms of LTP and endocannabinoid signal- ing) by cell adhesion molecules in CA1-PYR cell inputs and he brain’s “connectivity code” is thought to confer exquisite Tspecificity to brain circuits by dictating connectivity between different types of neurons. Although its existence has not yet Significance been conclusively demonstrated, synaptic cell adhesion mole- cules likely comprise a large part of such a code (1–4). Cell ad- Synapses functionally connect neurons in the brain and medi- hesion molecules are encoded by ∼2% of the genome and play ate information processing relevant to all aspects of life. central roles in all tissues. During brain development, precisely Among others, synaptic connections are enabled by cell adhe- matching presynaptic and postsynaptic cell adhesion molecule sion molecules, which connect presynaptic and postsynaptic interactions likely guide synapse formation and specify the membranes by binding to each other via the synaptic cleft. properties of synapses by activating signal transduction cascades Mammalian genomes express hundreds of cell adhesion mol- and recruiting scaffolding molecules, receptors, and active-zone ecules whose combinatorial utilization is thought to contribute proteins; in addition, such interactions could provide structural to the brain’s “connectivity code.” Such code could explain the support to synapses. However, the molecular mechanisms in- versatility of synapses as well as the logic of connectivity be- volved are not understood. Because cell adhesion molecule- tween cell types. Here, we used single-cell RNA sequencing to based interactions likely code for synapse specificity in a com- analyze the expression of cell adhesion molecules and other binatorial fashion (2, 3), an important step toward gaining insight signaling proteins in defined cell types, and found developmental into these molecular mechanisms is to eliminate nonfunctional patterns that potentially identify relevant elements of the possibilities, which—to a great extent—can be done by examin- connectivity code. ing cell type-specific expression of these molecules. If cell adhesion molecules dictate synapse properties, then Author contributions: C.F., S.D., J.A., R.C.M., S.R.Q., and T.C.S. designed research; C.F., S.D., such differences must be clearly present in interneuron and py- and J.A. performed research; C.F. analyzed data; and C.F. and T.C.S. wrote the paper. ramidal cell classes, which have diverse synaptic properties. For Reviewers: T.B., Tufts University; T.F.F., Institute of Experimental Medicine; and L.-H.T., Massachusetts Institute of Technology. example, within the hippocampal circuit, CA1 pyramidal (CA1- PYR) neurons receive convergent inputs from multiple, elec- The authors declare no conflict of interest. trophysiologically distinct inhibitory interneurons within the Data deposition: The sequence reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. hippocampus (5). Inhibitory inputs include synapses formed by GSE75386). fast-spiking (FS) and regular-spiking (RS) interneurons (ref. 6; 1To whom correspondence may be addressed. Email: [email protected] or tcs1@ for reviews, see refs. 7 and 8). We previously showed using sin- stanford.edu. gle-cell transcriptional profiling in FS parvalbumin (PV)- and RS This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cholecystokinin (CCK)-containing GABAergic interneurons that 1073/pnas.1610155113/-/DCSupplemental. E5222–E5231 | PNAS | Published online August 16, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1610155113 Downloaded by guest on September 27, 2021 outputs, and raised the question whether other synaptic prop- Results PNAS PLUS erties were also controlled by cell adhesion molecules. Because Cell Adhesion Molecules in the Hippocampus. As a starting point, we there are a large number of molecules with such potential, a examined the transcriptome of the entire hippocampus (Fig. 1A) logical step to this direction is the cell type-specific analysis of at five developmental stages (in triplicate at postnatal days P0, cell adhesion-related gene expression. P7, P14, P21, and P28). In these samples, the total number and Here, we combined electrophysiology and single-cell RNA se- distribution of expressed genes were similar (Fig. 1 B and C, and quencing (RNAseq) to identify cells in the pathway involving Fig. S1). To specifically analyze cell adhesion molecules, we hippocampal FS interneuron (FS-INT), RS-INT, CA1-PYR cells, created a comprehensive list of candidate genes involved in, or and subiculum RS-PYR and BS-PYR cells, and to analyze their related to, transsynaptic cell adhesion (collectively referred to as gene expression profiles. Our data represent an initial circuit-level CAMs, for candidate cell adhesion-related molecules). For this, single-cell RNAseq analysis from synaptically and electrophysio- we first considered all molecules with single transmembrane logically defined neurons. We find surprising differences in the domains (a general, although not unique property of membrane total number of expressed genes among neuron types and show surface adhesion molecules) and narrowed this list down to 406 that hippocampal neurons can be characterized by the expression genes based on preexisting data (Fig. S1C). This curated list of of two common, developmentally regulated gene networks com- candidate cell adhesion molecules includes proteins implicated prising shared cell adhesion and signaling molecules. Moreover, we in cell–cell signaling but excludes, for example, intracellular demonstrate that each type of electrophysiologically defined neu- signal