Development and characterization of a Gucy2d-cre mouse to selectively manipulate a subset of inhibitory spinal dorsal horn interneurons.

Recent transcriptomic studies identified Gucy2d (encoding guanylate cyclase D) as a highly enriched gene within inhibitory dynorphin interneurons in the mouse spinal dorsal horn. To facilitate investigations into the role of the Gucy2d+ population in somatosensation, Gucy2d-cre transgenic mice were created to permit chemogenetic or optogenetic manipulation of this subset of spinal neurons. Gucy2d-cre mice created via CRISPR/Cas9 genomic knock-in were bred to mice expressing a cre-dependent reporter (either tdTomato or Sun1.GFP fusion protein), and the resulting offspring were characterized. Surprisingly, a much wider population of spinal neurons was labeled by cre-dependent reporter expression than previous mRNA-based studies would suggest. Although the cre-dependent reporter expression faithfully labeled ~75% of cells expressing Gucy2d mRNA in the adult dorsal horn, it also labeled a substantial number of additional inhibitory neurons in which no Gucy2d or Pdyn mRNA was detected. Moreover, cre-dependent reporter was also expressed in various regions of the brain, including the spinal trigeminal nucleus, cerebellum, thalamus, somatosensory cortex, and anterior cingulate cortex. Injection of AAV-CAG-FLEX-tdTomato viral vector into adult Gucy2d-cre mice produced a similar pattern of cre-dependent reporter expression in the spinal cord and brain, which excludes the possibility that the unexpected reporter-labeling of cells in the deep dorsal horn and brain was due to transient Gucy2d expression during early stages of development. Collectively, these results suggest that Gucy2d is expressed in a wider population of cells than previously thought, albeit at levels low enough to avoid detection with commonly used mRNA-based assays. Therefore, it is unlikely that these Gucy2d-cre mice will permit selective manipulation of inhibitory signaling mediated by spinal dynorphin interneurons, but this novel cre driver line may nevertheless be useful to target a broader population of inhibitory spinal dorsal horn neurons.

Early-Life Iron Deficiency Persistently Alters Nociception in Developing Mice.

Clinical association studies have identified early-life iron deficiency (ID) as a risk factor for the development of chronic pain. While preclinical studies have shown that early-life ID persistently alters neuronal function in the central nervous system, a causal relationship between early-life ID and chronic pain has yet to be established. We sought to address this gap in knowledge by characterizing pain sensitivity in developing male and female C57Bl/6 mice that were exposed to dietary ID during early life. Dietary iron was reduced by ∼90% in dams between gestational day 14 and postnatal day (P)10, with dams fed an ingredient-matched, iron-sufficient diet serving as controls. While cutaneous mechanical and thermal withdrawal thresholds were not altered during the acute ID state at P10 and P21, ID mice were more sensitive to mechanical pressure at P21 independent of sex. During adulthood, when signs of ID had resolved, mechanical and thermal thresholds were similar between early-life ID and control groups, although male and female ID mice displayed increased thermal tolerance at an aversive (45 °C) temperature. Interestingly, while adult ID mice showed decreased formalin-induced nocifensive behaviors, they showed exacerbated mechanical hypersensitivity and increased paw guarding in response to hindpaw incision in both sexes. Collectively, these results suggest that early-life ID elicits persistent changes in nociceptive processing and appears capable of priming developing pain pathways. PERSPECTIVE: This study provides novel evidence that early-life ID evokes sex-independent effects on nociception in developing mice, including an exacerbation of postsurgical pain during adulthood. These findings represent a critical first step towards the long-term goal of improving health outcomes for pain patients with a prior history of ID.

Gucy2d selectively marks inhibitory dynorphin neurons in the spinal dorsal horn but is dispensable for pain and itch sensitivity.

INTRODUCTION: Inhibitory neurons in the spinal dorsal horn can be classified based on expression of neurochemical marker genes. However, these marker genes are often expressed throughout the central nervous system, which poses challenges for manipulating genetically identified spinal neurons without undesired off-target effects. OBJECTIVES: We investigated whether Gucy2d, previously identified as a highly selective marker of dynorphin-lineage neurons in the dorsal horn, is expressed in other locations within the adult mouse spinal cord, dorsal root ganglia (DRG), or brain. In addition, we sought to molecularly characterize Gucy2d-expressing dorsal horn neurons and investigate whether the disruption of Gucy2d gene expression affects sensitivity to itch or pain. METHODS: In situ hybridization experiments assessed Gucy2d mRNA expression in the adult mouse spinal cord, DRG, and brain, and its colocalization with Pax2, Bhlhb5, and Pde2a in dorsal horn neurons. Knockout mice lacking Gucy2d expression were compared with littermate controls to assess sensitivity to chloroquine-induced itch and dry skin-mediated chronic itch, as well as heat, cold, or mechanical stimuli. RESULTS: Gucy2d is selectively expressed in dynorphin-lineage neurons in lamina I-III of the adult mouse spinal cord but not in the brain or DRG. Spinal Gucy2d-expressing neurons are inhibitory neurons that also express the transcription factor Bhlhb5 and the cGMP-dependent phosphodiesterase Pde2a. Gucy2d knockout mice did not exhibit altered responses to itch or pain. CONCLUSIONS: The selective expression of Gucy2d within a subpopulation of inhibitory dorsal horn neurons may yield a means to selectively manipulate inhibitory signaling at the level of the spinal cord without effects on the brain.

A photoswitchable GPCR-based opsin for presynaptic inhibition.

Optical manipulations of genetically defined cell types have generated significant insights into the dynamics of neural circuits. While optogenetic activation has been relatively straightforward, rapid and reversible synaptic inhibition has proven more elusive. Here, we leveraged the natural ability of inhibitory presynaptic GPCRs to suppress synaptic transmission and characterize parapinopsin (PPO) as a GPCR-based opsin for terminal inhibition. PPO is a photoswitchable opsin that couples to Gi/o signaling cascades and is rapidly activated by pulsed blue light, switched off with amber light, and effective for repeated, prolonged, and reversible inhibition. PPO rapidly and reversibly inhibits glutamate, GABA, and dopamine release at presynaptic terminals. Furthermore, PPO alters reward behaviors in a time-locked and reversible manner in vivo. These results demonstrate that PPO fills a significant gap in the neuroscience toolkit for rapid and reversible synaptic inhibition and has broad utility for spatiotemporal control of inhibitory GPCR signaling cascades.

Implementing SAFE™ care: Evaluation of a geriatric model of care for real-world practice.

Systems Addressing Frail Elders (SAFETM) Care is a geriatric model of care that identifies high-risk hospitalized older adults, and provides targeted interprofessional interventions for risk factors associated with frailty. This post, mixed methods study sought to evaluate SAFETM Care implementation retrospectively at one public academic medical center and describe practical "real-world" considerations for implementation using the Consolidated Framework for Implementation Research (CFIR). In addition to barriers and facilitators, hidden characteristics to consider for implementation include initiating conditions, skills and experiences of implementers, interpersonal challenges, unique facilitators and barriers, surprising conditions, and threats to and requirements for sustainability. Implementation of SAFETM Care demonstrated effective adoption and implementation, but faced multiple threats that led to failed sustainability. The public sharing of these successes and failures will help implementers understand and make progress in adapting such important geriatric programs and quality improvement initiatives.

Cell type-specific modulation of sensory and affective components of itch in the periaqueductal gray.

Itch is a distinct aversive sensation that elicits a strong urge to scratch. Despite recent advances in our understanding of the peripheral basis of itch, we know very little regarding how central neural circuits modulate acute and chronic itch processing. Here we establish the causal contributions of defined periaqueductal gray (PAG) neuronal populations in itch modulation in mice. Chemogenetic manipulations demonstrate bidirectional modulation of scratching by neurons in the PAG. Fiber photometry studies show that activity of GABAergic and glutamatergic neurons in the PAG is modulated in an opposing manner during chloroquine-evoked scratching. Furthermore, activation of PAG GABAergic neurons or inhibition of glutamatergic neurons resulted in attenuation of scratching in both acute and chronic pruritis. Surprisingly, PAG GABAergic neurons, but not glutamatergic neurons, may encode the aversive component of itch. Thus, the PAG represents a neuromodulatory hub that regulates both the sensory and affective aspects of acute and chronic itch.

Enhanced hemangioblast generation and improved vascular repair and regeneration from embryonic stem cells by defined transcription factors.

The fetal liver kinase 1 (FLK-1)(+) hemangioblast can generate hematopoietic, endothelial, and smooth muscle cells (SMCs). ER71/ETV2, GATA2, and SCL form a core transcriptional network in hemangioblast development. Transient coexpression of these three factors during mesoderm formation stage in mouse embryonic stem cells (ESCs) robustly enhanced hemangioblast generation by activating bone morphogenetic protein (BMP) and FLK-1 signaling while inhibiting phosphatidylinositol 3-kinase, WNT signaling, and cardiac output. Moreover, etsrp, gata2, and scl inhibition converted hematopoietic field of the zebrafish anterior lateral plate mesoderm to cardiac. FLK-1(+) hemangioblasts generated by transient coexpression of the three factors (ER71-GATA2-SCL [EGS]-induced FLK-1(+)) effectively produced hematopoietic, endothelial, and SMCs in culture and in vivo. Importantly, EGS-induced FLK-1(+) hemangioblasts, when codelivered with mesenchymal stem cells as spheroids, were protected from apoptosis and generated functional endothelial cells and SMCs in ischemic mouse hindlimbs, resulting in improved blood perfusion and limb salvage. ESC-derived, EGS-induced FLK-1(+) hemangioblasts could provide an attractive cell source for future hematopoietic and vascular repair and regeneration.