Itch is an unpleasant sensation that provokes the urge to scratch. The sensation of itch can beinduced by distinct triggers including pruritogen (chemical itch) or tactile stimuli (mechanical itch). Chemical and mechanical itch, due to distinct nature of the stimuli, seem mediated by remarkably different peripheral and central neural circuitries (Bourane et al. 2015; Acton et al. 2019; Pan et al. 2019; Chen et al. 2020). A cohort of molecularly defined sensory and spinal neuron populations that relay chemical itch has been characterized, while very little is known about those that relay mechanical itch. Scratching is usually the immediate response induced by itch in human and animals to remove dangerous pruritic agents. However, when itch becomes prolonged or chronic, affective motivational components of itch, such as negative valence and aversive memory induced by itch, form and more complicated protective behavior is recruited to start itch-relief, which require the involvement of the supraspinal itch-relaying regions through ascending and descending itch pathways. It has been reported that chemical itch sensation is potentially relayed by spinal projection neurons that express Tacr1 (as known as NK1R) to the parabrachial nuclei (Carsten et al. 2010; Acton et al. 2019). However, it is still not known what supraspinal structures encode mechanical itch responses and how mechanical itch information is transmitted to these supraspinal regions through ascending pathways. By focusing on these questions in my thesis project, I have tried to answer these questions by addressing three more specific issues: 1. What is the identity of supraspinal region that relays mechanical itch? 2. What is the molecular identity of the spinal projection neurons that relay mechanical itch information and who do they connect to? 3. What is the molecular identity of the neurons in the supraspinal region that are contacted by the spinal projection neurons that transmit mechanical itch and are they also necessary for itch-induced scratching? In the first part of my thesis, I identified the parabrachial nucleus (PBN), located in the dorsolateral pons area of the brainstem, as a crucial relay hub for mechanical itch. Chemogenetic silencing of the PBN neurons suppresses mechanical itch sensitivities under both acute and chronic conditions in mice. In the second part of my thesis, I have characterized a spinal neuron population that is selectively targeted by CalcrlCre and Lbx1FlpO (hereafter referred to as CalcrlLbx1 neurons) that have a dedicated projection to the PBN. Genetic ablation or chemogenetic silencing of this neuron population in the spinal cord suppresses mechanical itch sensitivities under baseline, acute as well as chronic itch conditions. By contrast, chemogenetic activation of the CalcrlLbx1 population dramatically sensitizes mechanical itch in mice. A similar but lesser extensive suppression of mechanical itch sensitivity was observed when the spinoparabrachial projections of CalcrlLbx1 neurons were selectively silenced. In the third part of my thesis, I have shown that PBN neurons that express FoxP2 function as a postsynaptic partner of spinal CalcrlLbx1 neurons and are necessary for relaying and expressing mechanical itch. In vivo calcium imaging by fiber photometry showed FoxP2PBN neurons are tuned to respond to mechanical itch stimulation under baseline, acute and chronic itch conditions. Chemogenetic silencing of these neurons in the PBN suppresses mechanical itch sensitivity in all conditions. In summary, my thesis project identified and characterized a novel mechanical itch-relaying spinoparabrachial pathway featured by the Calcrl-FoxP2 functional connection.

Movement is a fundamental behavior that is generated and patterned by premotor neural networks that reside in the ventral spinal cord of vertebrates. Despite ongoing investigations in multiple animal models, including humans, non-human primates, cats and rodents, identifying the functional neuron cell types that shape various motor tasks has proved difficult. One of the key issues in the field is the criteria by which neuronal cell types are characterized and defined. This dissertation describes my work tackling this problem using a comprehensive anatomical approach aimed at identifying neuronal subsets within major classes of ipsilateral premotor spinal interneurons. The first chapter provides a historical background of spinal motor system research developed from the beginning of the 20th century that summarizes our current knowledge of motor circuits in the spinal cord and the contribution that this dissertation makes to bridging the gaps that exist in our understanding of premotor interneuron cell types. The second chapter describes the anatomical approach I pursued, which is based on spatial distribution, connectivity and morphology to identify potential groups of functional premotor interneuron cell types.In the third chapter I discuss the relevance of these findings, by highlighting how the strategy used in this work can be applied to other spinal cord populations and used to lay the groundwork to build an anatomical reference for motor functional studies, including electrophysiology and behavioral assays. This work provides an important and necessary framework to understand the complicated logic governing the organization of motor networks in the vertebrate spinal cord.