This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. A link to any extended data will be provided when the final version is posted online. Research Articles: Systems/Circuits Identification of two classes of somatosensory neurons that display resistance to retrograde infection by rabies virus Gioele W. Albisetti1, Alexander Ghanem2, Edmund Foster1, Karl-Klaus Conzelmann2, Hanns Ulrich Zeilhofer1,3 and Hendrik Wildner1 1Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland 2Max von Pettenkofer Institute & Gene Center, Virology, Faculty of Medicine, LMU München, Feodor-Lynen-Str. 25, 81377 München, Germany 3Institute of Pharmaceutical Sciences, Swiss Federal Institute (ETH) Zurich, Wolfgang-Pauli Strasse, CH-8090 Zürich, Switzerland DOI: 10.1523/JNEUROSCI.1277-17.2017 Received: 9 May 2017 Revised: 29 August 2017 Accepted: 11 September 2017 Published: 26 September 2017 Author contributions: GA performed virus injections and performed and analyzed morphological experiments. K.K.C. and A.G. produced and provided the majority of pseudotyped G-deleted rabies viruses used in this study. EF performed virus injections. HW analyzed experiments. H.W. and H.U.Z. designed experiments and wrote the manuscript. All authors have commented on the manuscript. Conflict of Interest: The authors declare no competing financial interests. This work was supported by an ERC Advanced Investigator Grant (DHISP 250128), a grant from the Swiss National Research Foundation (156393) and a Wellcome Trust Collaborative Award in Science (200183/Z/15/ Z) (to H.U.Z.). HW was supported by the Olga Mayenfisch foundation. KKC and AG were supported by DFG SFB870 (KKC) and LMUexcellent and LMU WiFoMed (AG). Correspondence: Dr Hendrik Wildner, Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland, Tel. +41 44 63 55938, [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.1277-17.2017 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2017 Albisetti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 Identification of two classes of somatosensory neurons that display resistance 2 to retrograde infection by rabies virus 3 DRG neuron subsets resistant to rabies infection 4 1Gioele W. Albisetti, 2Alexander Ghanem, 1Edmund Foster 2Karl-Klaus Conzelmann, 1,3Hanns Ulrich 5 Zeilhofer, 1Hendrik Wildner 6 1Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH-8057 7 Zürich, Switzerland 8 2Max von Pettenkofer Institute & Gene Center, Virology, Faculty of Medicine, LMU München, Feodor- 9 Lynen-Str. 25, 81377 München, Germany3Institute of Pharmaceutical Sciences, Swiss Federal 10 Institute (ETH) Zurich, Wolfgang-Pauli Strasse, CH-8090 Zürich, Switzerland 11 12 Correspondence: Dr Hendrik Wildner 13 Institute of Pharmacology and Toxicology 14 University of Zurich 15 Winterthurerstrasse 190 16 CH-8057 Zurich 17 Switzerland 18 Tel. +41 44 63 55938 19 [email protected] 20 21 Number of pages: 16 22 Number of figures: 7 23 Number of words for Abstract (161), Introduction (622), and Discussion (1386) 24 Conflict of Interest: There is no conflict of interest 25 Acknowledgements: 26 This work was supported by an ERC Advanced Investigator Grant (DHISP 250128), a grant from the 27 Swiss National Research Foundation (156393) and a Wellcome Trust Collaborative Award in Science 28 (200183/Z/15/Z) (to H.U.Z.). HW was supported by the Olga Mayenfisch foundation. KKC and AG 29 were supported by DFG SFB870 (KKC) and LMUexcellent and LMU WiFoMed (AG). 30 - 1 - 31 Abstract 32 Glycoprotein-deleted ('G) rabies virus-mediated monosynaptic tracing has become a standard 33 method for neuronal circuit mapping, and is applied to virtually all parts of the rodent nervous 34 system including the spinal cord and primary sensory neurons. Here we identified two classes of 35 unmyelinated sensory neurons (non-peptidergic/NP- and C-LTMR/TH neurons) that are resistant to 36 direct and transsynaptic infection from the spinal cord with rabies viruses carrying glycoproteins in 37 their envelopes that are routinely used for infection of CNS neurons (SAD-G or N2C-G). However, the 38 same neurons were susceptible to infection with EnvA-pseudotyped rabies virus in TVA transgenic 39 mice, indicating that resistance to retrograde infection was due to impaired virus adsorption rather 40 than to deficits in subsequent steps of infection. These results demonstrate an important limitation 41 of rabies virus-based retrograde tracing of sensory neurons in adult mice, and may help to better 42 understand the molecular machinery required for rabies virus spread in the nervous system. In this 43 study mice of both sexes were used. 44 45 Significance Statement 46 In order to understand the neuronal bases of behavior it is important to identify the underlying 47 neural circuitry. Rabies virus based monosynaptic tracing has been used to identify neuronal circuits 48 in various parts of the nervous system. This has included connections between peripheral sensory 49 neurons and their spinal targets, which form the first synapse in the somatosensory pathway. Here 50 we demonstrate that two classes of unmyelinated sensory neurons, which account for more than 51 40% of DRG neurons, display resistance to rabies infection. Our results are therefore critical for 52 interpreting monosynaptic rabies-based tracing in the sensory system. In addition, identification of 53 rabies resistant neurons might provide a means for future studies addressing rabies pathobiology. - 2 - 54 Introduction 55 Rabies virus infection of different parts of the nervous system has been used for more than 20 years 56 to identify functional networks of synaptically connected neurons (Ugolini, 1995, 2008). Unequivocal 57 identification of monosynaptically connected neurons (monosynaptic retrograde tracing) has become 58 possible through deletion of the glycoprotein (G-protein) gene from the genome of the rabies 59 vaccine strain SAD B19 and inclusion of fluorescent reporter genes such as GFP (Wickersham et al., 60 2007) for review see (Callaway and Luo, 2015; Ghanem and Conzelmann, 2016). Deletion of the G- 61 protein coding sequence renders the virus non-infectious, while inclusion of a GFP expression 62 cassette allows fast identification of infected neurons. Infectivity of the G-deleted rabies virus can be 63 restored in vivo through trans-complementation of the virus with a G-protein expressed either from 64 a plasmid, a helper virus or a mouse transgene. This complementation enables monosynaptic 65 retrograde spread and neurons providing direct synaptic input to the primary infected cells can then 66 be identified by the expression of the reporter gene. This powerful approach has been rapidly 67 adapted and is now widely used for circuit mapping throughout the nervous system (for review see 68 (Callaway and Luo, 2015)). 69 We and others have used this approach to identify sensory neuron subtypes providing input to 70 defined types of second order neurons in the spinal dorsal horn. The cell bodies of these sensory 71 neurons are located in the dorsal root ganglia (DRG) from where they send axons to the periphery 72 and to the spinal dorsal horn. Their peripheral terminals function as detectors of mechanical, 73 thermal, proprioceptive and noxious stimuli, while the central (spinal) terminals of these neurons 74 form synaptic contacts with second order neurons. A comprehensive characterization of the 75 connections between DRG neurons and spinal neurons is crucial for our understanding of how 76 different sensory modalities are processed, modified and finally relayed to supraspinal sites where 77 they are perceived. 78 Primary sensory nerve fibers can be subdivided broadly into myelinated (AE and AG) fibers and 79 unmyelinated C-fibers. Myelinated AE fibers terminate predominantly in the deep dorsal horn 80 (lamina III and deeper), while the terminals of unmyelinated C-fibers are found almost exclusively in 81 laminae I and II. To identify the connections formed between sensory neurons and genetically 82 defined spinal neuron populations, several groups have employed monosynaptic rabies virus-based 83 tracing methods (Bourane et al., 2015b; Bourane et al., 2015a; Foster et al., 2015; Francois et al., 84 2017; Sun et al., 2017). These reports provide strong evidence for direct synaptic input from 85 myelinated sensory fibers. However, only one study has reported retrograde infection of 86 unmyelinated non-peptidergic (equivalent to the isolectin B4 [IB4] binding population of sensory 87 neurons) or unmyelinated low threshold mechanoreceptive (c-LTMR / TH+) sensory neurons. This is 88 highly unexpected as IB4+ neurons account for about 30% and TH+ for about 14% of all sensory 89 neurons innervating the lumbar spinal dorsal horn. 90 To systematically investigate the ability of G-deleted rabies virus to infect all classes of DRG neurons, 91 we directly injected G-deleted rabies virus pseudotyped with SAD-G