Spinal transmission of pruritoceptive (itch) signals requires transneuronal signaling by gastrin-releasing peptide (GRP) produced by a subpopulation of dorsal horn excitatory interneurons. These neurons also express the glutamatergic marker vGluT2, raising the question of why glutamate alone is insufficient for spinal itch relay. Using optogenetics together with slice electrophysiology and mouse behavior, we demonstrate that baseline synaptic coupling between GRP and GRP receptor (GRPR) neurons is too weak for suprathreshold excitation. Only when we mimicked the endogenous firing of GRP neurons and stimulated them repetitively to fire bursts of action potentials did GRPR neurons depolarize progressively and become excitable by GRP neurons. GRPR but not glutamate receptor antagonism prevented this action. Provoking itchlike behavior by optogenetic activation of spinal GRP neurons required similar stimulation paradigms. These results establish a spinal gating mechanism for itch that requires sustained repetitive activity of presynaptic GRP neurons and postsynaptic GRP signaling to drive GRPR neuron output.

Gastrin-releasing peptide (GRP) is a spinal itch transmitter expressed by a small population of dorsal horn interneurons (GRP neurons). The contribution of these neurons to spinal itch relay is still only incompletely understood, and their potential contribution to painrelated behaviors remains controversial. Here, we have addressed this question in a series of experiments performed in GRP::cre and GRP::eGFP transgenic male mice. We combined behavioral tests with neuronal circuit tracing, morphology, chemogenetics, optogenetics, and electrophysiology to obtain a more comprehensive picture. We found that GRP neurons form a rather homogeneous population of central cell-like excitatory neurons located in lamina II of the superficial dorsal horn. Multicolor high-resolution confocal microscopy and optogenetic experiments demonstrated that GRP neurons receive direct input from MrgprA3-positive pruritoceptors. Anterograde HSV-based neuronal tracing initiated from GRP neurons revealed ascending polysynaptic projections to distinct areas and nuclei in the brainstem, midbrain, thalamus, and the somatosensory cortex. Spinally restricted ablation of GRP neurons reduced itch-related behaviors to different pruritogens, whereas their chemogenetic excitation elicited itch-like behaviors and facilitated responses to several pruritogens. By contrast, responses to painful stimuli remained unaltered. These data confirm a critical role of dorsal horn GRP neurons in spinal itch transmission but do not support a role in pain.

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.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.