Identification of Two Classes of Somatosensory Neurons That Display Resistance to Retrograde Infection by Rabies Virus

Abstract

Glycoprotein-deleted rabies virus-mediated monosynaptic tracing has become a standard method for neuronal circuit mapping, and is applied to virtually all parts of the rodent nervous system, including the spinal cord and primary sensory neurons. Here we identified two classes of unmyelinated sensory neurons (nonpeptidergic and C-fiber low-threshold mechanoreceptor neurons) resistant to direct and trans-synaptic infection from the spinal cord with rabies viruses that carry glycoproteins in their envelopes and that are routinely used for infection of CNS neurons (SAD-G and N2C-G). However, the same neurons were susceptible to infection with EnvA-pseudotyped rabies virus in tumor virus A receptor transgenic mice, indicating that resistance to retrograde infection was due to impaired virus adsorption rather than to deficits in subsequent steps of infection. These results demonstrate an important limitation of rabies virus-based retrograde tracing of sensory neurons in adult mice, and may help to better understand the molecular machinery required for rabies virus spread in the nervous system. In this study, mice of both sexes were used.SIGNIFICANCE STATEMENT To understand the neuronal bases of behavior, it is important to identify the underlying neural circuitry. Rabies virus-based monosynaptic tracing has been used to identify neuronal circuits in various parts of the nervous system. This has included connections between peripheral sensory neurons and their spinal targets. These connections form the first synapse in the somatosensory pathway. Here we demonstrate that two classes of unmyelinated sensory neurons, which account for >40% of dorsal root ganglia neurons, display resistance to rabies infection. Our results are therefore critical for interpreting monosynaptic rabies-based tracing in the sensory system. In addition, identification of rabies-resistant neurons might provide a means for future studies addressing rabies pathobiology.

Expression of green fluorescent protein defines a specific population of lamina II excitatory interneurons in the GRP::eGFP mouse

“…The remainder may include contributions from other classes of primary afferent (e.g. myelinated LTMRs 21 , thermoreceptors or those that express MrgB4 52 ) as well as from descending sources such as the corticospinal tract 27 .…”

“…In addition, mice lacking GRPR, or those in which GRPR-expressing dorsal horn neurons are ablated, show reduced responses in several itch models 7 , 8 , 12 , 13 . Excitatory interneurons within the SDH provide a source of GRP 7 , 14 – 16 , and several studies have investigated the role of these cells by using BAC transgenic mouse lines from the GENSAT project 17 in which either enhanced green fluorescent protein (eGFP) 7 , 14 , 15 , 18 – 20 or Cre recombinase 18 – 21 is expressed under control of the Grp promoter (GRP::eGFP and GRP::Cre, respectively). We have reported that the eGFP-positive cells in the GRP::eGFP line are all excitatory, accounting for ~ 15% of the excitatory interneurons in laminae I-II 4 , 14 , and that they are largely separate from populations defined by the expression of five other peptides: cholecystokinin (CCK), neurotensin, neurokinin B (NKB), neuropeptide FF (NPFF) and substance P (SP) 3 , 4 , 22 , 23 .…”

Retrograde axonal transport of rabies virus is unaffected by interferon treatment but blocked by emetine locally in axons

“…To date, sensory dorsal root ganglia (DRG) or ventral spinal cord neurons have been used for RABV axonal transport studies. However, unlike SCG, spinal cord neurons exhibit limited axonal projections in compartmented cultures [ 12 ], and DRG are heterogenous with subpopulations of unmyelinated neurons that display resistance to RABV infection [ 38 ]. We established an ex vivo model of RABV infection in compartmented SCG neurons to facilitate high resolution imaging of RABV axon transport as well as concurrent biochemical analyses of RABV-infected axons.…”

Dorsal Horn Gastrin-Releasing Peptide Expressing Neurons Transmit Spinal Itch But Not Pain Signals

“…Intraspinal virus injections were made in isoflurane-anesthetized mice using a motorized stereotaxic frame (David Kopf Instruments and Neurostar). Three separate unilateral injections of 300 nl each were performed at a depth of 300 m using glass micropipettes connected to a PHD ultra nanomite syringe pump (Harvard Apparatus) as described previously (Albisetti et al, 2017;Haenraets et al, 2018). The following viruses were used: for chemogenetic activation, AAV1.hSyn.flex.…”