RESUMEN
Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. However, current methods for chronically implanting probes present steep trade-offs between cost, ease of use, size, adaptability, and long-term stability. This protocol introduces a novel chronic probe implant system for mice called the DREAM (Dynamic, Recoverable, Economical, Adaptable, and Modular), designed to overcome the trade-offs associated with currently available options. The system provides a lightweight, modular and cost-effective solution with standardized hardware elements that can be combined and implanted in straightforward steps and explanted safely for recovery and multiple reuse of probes, significantly reducing experimental costs. The DREAM implant system integrates three hardware modules: (1) a microdrive that can carry all standard silicon probes, allowing experimenters to adjust recording depth across a travel distance of up to 7 mm; (2) a three-dimensional (3D)-printable, open-source design for a wearable Faraday cage covered in copper mesh for electrical shielding, impact protection, and connector placement, and (3) a miniaturized head-fixation system for improved animal welfare and ease of use. The corresponding surgery protocol was optimized for speed (total duration: 2 h), probe safety, and animal welfare. The implants had minimal impact on animals' behavioral repertoire, were easily applicable in freely moving and head-fixed contexts, and delivered clearly identifiable spike waveforms and healthy neuronal responses for weeks of post-implant data collection. Infections and other surgery complications were extremely rare. As such, the DREAM implant system is a versatile, cost-effective solution for chronic electrophysiology in mice, enhancing animal well-being, and enabling more ethologically sound experiments. Its design simplifies experimental procedures across various research needs, increasing accessibility of chronic electrophysiology in rodents to a wide range of research labs.
Asunto(s)
Electrodos Implantados , Electrofisiología , Animales , Ratones , Electrofisiología/instrumentación , Electrofisiología/métodos , Conducta Animal/fisiología , Fenómenos Electrofisiológicos , Análisis Costo-BeneficioRESUMEN
Somatosensation, the detection and transduction of external and internal stimuli such as temperature or mechanical force, is vital to sustaining our bodily integrity. But still, some of the mechanisms of distinct stimuli detection and transduction are not entirely understood, especially when noxious perception turns into chronic pain. Over the past decade major progress has increased our understanding in areas such as mechanotransduction or sensory neuron classification. However, it is in particular the access to human pluripotent stem cells and the possibility of generating and studying human sensory neurons that has enriched the somatosensory research field. Based on our previous work, we describe here the generation of human stem cell-derived nociceptor-like cells. We show that by varying the differentiation strategy, we can produce different nociceptive subpopulations with different responsiveness to nociceptive stimuli such as capsaicin. Functional as well as deep sequencing analysis demonstrated that one protocol in particular allowed the generation of a mechano-nociceptive sensory neuron population, homogeneously expressing TRPV1. Accordingly, we find the cells to homogenously respond to capsaicin, to become sensitized upon inflammatory stimuli, and to respond to temperature stimulation. The efficient and homogenous generation of these neurons make them an ideal translational tool to study mechanisms of sensitization, also in the context of chronic pain.
Asunto(s)
Capsaicina , Dolor Crónico , Capsaicina/farmacología , Humanos , Mecanotransducción Celular , Células Receptoras Sensoriales/metabolismo , Células Madre/metabolismo , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
High serum concentrations of TNF-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor protein family, are found in patients with increased BMI and serum lipid levels. In a model of murine obesity, both the expression of TRAIL and its receptor (TRAIL-R) is elevated in adipose tissue. Accordingly, TRAIL has been proposed as an important mediator of adipose tissue inflammation and obesity-associated diseases. The aim of this study was to investigate if TRAIL regulates inflammatory processes at the level of the adipocyte. Using human Simpson-Golabi-Behmel syndrome (SGBS) cells as a model system, we found that TRAIL induces an inflammatory response in both preadipocytes and adipocytes. It stimulates the expression of interleukin 6 (IL-6), interleukin 8 (IL-8) as well as the chemokines monocyte chemoattractant protein-1 (MCP-1) and chemokine C-C motif ligand 20 (CCL-20) in a time- and dose-dependent manner. By using small molecule inhibitors, we found that both the NFκB and the ERK1/2 pathway are crucial for mediating the effect of TRAIL. Taken together, we identified a novel pro-inflammatory function of TRAIL in human adipocytes. Our findings suggest that targeting the TRAIL/TRAIL-R system might be a useful strategy to tackle obesity-associated adipose tissue inflammation.
Asunto(s)
Adipocitos/efectos de los fármacos , Inflamación/fisiopatología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Adulto , Arritmias Cardíacas , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocina CCL20 , Enfermedades Genéticas Ligadas al Cromosoma X , Gigantismo , Cardiopatías Congénitas , Humanos , Discapacidad Intelectual , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Transducción de SeñalRESUMEN
Tumor necrosis factor-α (TNFα) and other ligands of the TNF superfamily are potent regulators of adipose tissue metabolism and play a crucial role in the obesity-induced inflammation of adipose tissue. Adipose tissue expression levels of TRAIL (TNF-related apoptosis-inducing ligand) and its receptor were shown to be upregulated by overfeeding and decreased by fasting in mice. In the present study we aimed to elucidate the impact of TRAIL on adipogenesis. To this end, human Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes as well as stromal-vascular cells isolated from human white adipose tissue were used as model systems. Human recombinant TRAIL inhibited adipogenic differentiation in a dose-dependent manner. It activated the cleavage of caspase-8 and -3, which in turn resulted in a downregulation of the key adipogenic transcription factors C/EBPα, C/EBPδ, and PPARγ. The effect was completely blocked by pharmacological or genetic inhibition of caspases. Taken together we discovered a so far unrecognized function of TRAIL in the regulation of adipogenesis. Targeting the TRAIL/TRAIL receptor system might provide a novel strategy to interfere with adipose tissue homeostasis.