Graphical abstract

13The passage of nociceptive information is relayed through the spinal cord dorsal horn, a critical area in 14 sensory processing. The neuronal circuits in this region that underpin sensory perception must be clarified to 15 better understand how dysfunction can lead to pathological pain. This study used an optogenetic approach to 16 selectively activate neurons that contain the calcium-binding protein calretinin (CR). We show that CR + 17 interneurons form an interconnected network that can initiate and sustain enhanced excitatory signaling, and 18 directly relays signals to lamina I projection neurons. In vivo photoactivation of CR + interneurons resulted in 19 a significant nocifensive behavior that was morphine sensitive and cause a conditioned place aversion. 20Furthermore, halorhodopsin-mediated inhibition of CR + interneurons elevated sensory thresholds. These 21 results suggest that neuronal circuits in the superficial dorsal horn that involve excitatory CR + neurons are 22 important for the generation and amplification of pain, and identify these interneurons as a future analgesic 23 target. 24VGLUT3 relays low threshold input to CR + neurons (Peirs et al., 2015), however, a major limitation remains 53 the lack of detailed information on the postsynaptic circuits engaged by the CR + population to drive 54 behavioral responses. 56Here, we take an optogenetic approach to resolve the neuronal circuits excited by CR + neurons in laminae I 57 and II, and determine the functional significance of these neurons for sensory processing and perception. The 58 postsynaptic targets of CR + neurons were identified combining optogenetic stimulation with in vitro 59 electrophysiology, and also producing activation maps in anesthetized animals. This identified somatostatin + 60 neurons, neurokinin 1 receptor positive spinoparabrachial projection neurons, and CR + neurons themselves 61 among recipient populations for CR + input. Together, these populations form a highly integrated excitatory 62 network that is able to amplify dorsal horn circuit activity including downstream neural targets. Using in vivo 63 optogenetic stimulation in awake and behaving animals we were also able to show that spinal activation of 64 CR + neurons induces nocifensive behavior. 65Results 66 Optogenetic activation of spinal CR + neurons 67To study spinal CR + neuron connectivity and function in sensory processing, CR-Cre mice (Cr-IRES-Cre) 68were crossed with loxP-flanked-ChR2-eYFP mice (Ai32) to generate offspring where ChR2 was expressed 69 in CR + neurons (CR cre ;Ai32). These mice exhibited characteristic ChR2-eYFP expression in neurons and 70 fibers located in the superficial DH of the spinal cord forming a plexus that was concentrated in lamina IIo 71(Supplementary Figure 1A). This is consistent with the known pattern of CR expression in this spinal cord 72 region (Lu and Perl, 2003). Comparison with immunolabelling for CR confirmed ChR2-eYFP expression was 73 highly localized to the CR + population with 78.3 ± 4 % (St. Dev.) of CR + neur...