Nociceptive information is relayed through the spinal cord dorsal horn, a critical area in sensory processing. The neuronal circuits in this region that underpin sensory perception must be clarified to better understand how dysfunction can lead to pathological pain. This study used an optogenetic approach to selectively activate spinal interneurons that express the calcium-binding protein calretinin (CR). We show that these interneurons form an interconnected network that can initiate and sustain enhanced excitatory signaling, and directly relay signals to lamina I projection neurons. Photoactivation of CR interneurons in vivo resulted in a significant nocifensive behavior that was morphine sensitive, caused a conditioned place aversion, and was enhanced by spared nerve injury. Furthermore, halorhodopsin-mediated inhibition of these interneurons elevated sensory thresholds. Our results suggest that dorsal horn circuits that involve excitatory CR neurons are important for the generation and amplification of pain and identify these interneurons as a future analgesic target.

HighlightsCalretinin is differentially expressed among excitatory interneurons in the superficial dorsal horn of mouse spinal cord.Calretinin is present in many excitatory interneurons that express neurokinin B, substance P or gastrin-releasing peptide.Consistent with recent transcriptomic data, we find that most inhibitory substance P-expressing neurons contain calretinin.

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...

Background The purpose of this study is to undertake a comprehensive systematic review to describe multi-level factors (barriers and facilitators) that may influence the implementation of low-dose chest computed tomography (LDCT) for lung cancer screening in the United States. Methods Systematic literature searches were performed using six online databases and citation indexes for peer-reviewed studies, for articles published from 2013–2021. Studies were classified into three perspectives, based on the study’s unit of analysis: system, healthcare provider, and patient. Barriers and facilitators identified for each study included in our final review were then coded and categorized using the Consolidate Framework for Implementation Research (CFIR) domains. Results At the system level, the two most common constructs were external policy and incentives, and executing the implementation process. At the provider level, the most common constructs were evidence strength and quality of the intervention characteristics, patient needs and resources, implementation climate, and individual’s knowledge and beliefs about the intervention. At the patient-level, the most common constructs were patient needs and resources, individual’s knowledge and beliefs about the intervention, and engaging in the implementation process. These constructs can act both as facilitators or barriers to lung cancer screening implementation. Conclusions Applying the CFIR domains and constructs to understand and specify factors facilitating uptake of lung cancer screening, as well as cataloguing the lessons learned from previous efforts, help to inform the development and implementation processes of lung cancer screening programs in the community setting. Registration PROSPERO, CRD42021247677

Neurons located in dorsal root ganglia (DRG) are crucial for transmitting peripheral sensations such as proprioception, touch, temperature, and nociception to the spinal cord before propagating these signals to higher brain structures. To date, difficulty in identifying modality-specific DRG neurons has limited our ability to study specific populations in detail. As the calcium-binding protein parvalbumin (PV) is a neurochemical marker for proprioceptive DRG cells we used a transgenic mouse line expressing green fluorescent protein (GFP) in PV positive DRGs, to study the functional and molecular properties of putative proprioceptive neurons. Immunolabeled DRGs showed a 100% overlap between GFP positive (GFP+) and PV positive cells, confirming the PVeGFP mouse accurately labeled PV neurons. Targeted patch-clamp recording from isolated GFP+ and GFP negative (GFP−) neurons showed the passive membrane properties of the two groups were similar, however, their active properties differed markedly. All GFP+ neurons fired a single spike in response to sustained current injection and their action potentials (APs) had faster rise times, lower thresholds and shorter half widths. A hyperpolarization-activated current (I h) was observed in all GFP+ neurons but was infrequently noted in the GFP− population (100% vs. 11%). For GFP+ neurons, I h activation rates varied markedly, suggesting differences in the underlying hyperpolarization-activated cyclic nucleotide-gated channel (HCN) subunit expression responsible for the current kinetics. Furthermore, quantitative polymerase chain reaction (qPCR) showed the HCN subunits 2, 1, and 4 mRNA (in that order) was more abundant in GFP+ neurons, while HCN 3 was more highly expressed in GFP− neurons. Likewise, immunolabeling confirmed HCN 1, 2, and 4 protein expression in GFP+ neurons. In summary, certain functional properties of GFP+ and GFP− cells differ markedly, providing evidence for modality-specific signaling between the two groups. However,