P2Y receptors in the mammalian nervous system: pharmacology, ligands and therapeutic potential

P2Y receptors for extracellular nucleotides are coupled to activation of a variety of G proteins and stimulate diverse intracellular signaling pathways that regulate functions of cell types that comprise the central nervous system (CNS). There are 8 different subtypes of P2Y receptor expressed in cells of the CNS that are activated by a select group of nucleotide agonists. Here, the agonist selectivity of these 8 P2Y receptor subtypes is reviewed with an emphasis on synthetic agonists with high potency and resistance to degradation by extracellular nucleotidases that have potential applications as therapeutic agents. In addition, the recent identification of a wide variety of subtype-selective antagonists is discussed, since these compounds are critical for discerning cellular responses mediated by activation of individual P2Y receptor subtypes. The functional expression of P2Y receptor subtypes in cells that comprise the CNS is also reviewed and the role of each subtype in the regulation of physiological and pathophysiological responses is considered. Other topics include the role of P2Y receptors in the regulation of blood-brain barrier integrity and potential interactions between different P2Y receptor subtypes that likely impact tissue responses to extracellular nucleotides in the CNS. Overall, current research suggests that P2Y receptors in the CNS regulate repair mechanisms that are triggered by tissue damage, inflammation and disease and thus P2Y receptors represent promising targets for the treatment of neurodegenerative diseases

Up-regulation of the P2Y2 receptor by cytokines in neuronal cells

Abstract only availableAlzheimer's Disease (AD) is characterized by inflammation and neurodegeneration in the brain due to the presence of extracellular amyloid beta (A β) plaques and neurofibrillary tangles. Microglial and astrocyte cells associated with these plaques and tangles have been shown to release cytokines in AD patients, which have a proinflammatory effect on the brain. The P2Y2 receptor (P2Y2R) is a receptor protein that is up-regulated in response to damage or stress in a variety of tissues, including blood vessels and salivary gland epithelium. Recently our laboratory has shown that activation of the P2Y2R enhances α -secretase-dependent amyloid precursor protein (APP) processing. APP is proteolytically processed by β - and γ -secretases to release neurodegenerative A β. Alternatively, APP can be cleaved within the A β domain by α -secretase releasing the non-amyloidogenic product, sAPP α, which has been shown to have neuroprotective properties. Primary neurons have low P2Y2R expression, however, it has been demonstrated that cytokines up-regulate P2Y2R in smooth muscle cells. Therefore, this study will explore if cytokines up-regulate P2Y2R expression in primary rat neurons and in SH-SY5Y human neuroblastoma cells. Primary rat neurons and SH-SY5Y human neuroblastoma cells were plated on glass cover slips 24 or 48 hours with individual treatment, or a combination of, human interleukin-1 β (IL1- β), tumor necrosis factor α (TNF α), and interferon γ (IF γ). P2Y2R activity was measured by increases in intracellular calcium concentration ([Ca2+]i ) in response to the P2Y2R agonist UTP. Results support the hypothesis that P2Y2R is up-regulated by cytokines in neuronal cells. Furthermore, real-time PCR results indicate a two-fold increase in P2Y2R mRNA after cytokine treatment. Therefore, activation of the up-regulated P2Y2R in stressed neurons generates a neuroprotective (sAPP α) rather than neurodegenerative (A β) peptide. These results could have a substantial impact on the understanding and treatment of neurological disorders such as AD.Life Sciences Undergraduate Research Opportunity Progra

How Many Squirrels Are in the Shrubs? A Lesson Plan for Comparing Methods for Population Estimation

Estimating the population sizes of animals is a key skill for any student interested in ecology, conservation, or management. However, counting animals in natural habitats is difficult, and the many techniques that exist each rely on assumptions that can bias results. Most wildlife courses teach one or two of these methods, but rarely are students given an opportunity to compare approaches and explore how underlying assumptions affect the accuracy of estimates. Here, we describe a hands-on activity in which students estimate the size of a single population of animals using multiple methods: strip censuses, scat counts, and camera traps. They then compare the estimates and evaluate how the assumptions of each model (e.g., random use of habitats and animal behavior) bias the results. Finally, students submit their data to a national database that aggregates observations across multiple institutions as part of Squirrel-Net (http://squirrel-net.org). They can then analyze the national dataset, permitting exploration of these questions across a broader variety of habitats and species than would be possible at any single institution. Extensions of this activity guide students to enumerate the advantages and disadvantages of each method in different contexts and to select the most appropriate method for a given scenario. This activity and the database focus on estimating population sizes of squirrels, which are diurnal, charismatic, easily identified, and present in a wide range of habitats (including many campuses), but the same methods could be broadly used for other terrestrial species, including birds, amphibians, reptiles, or invertebrates

Squirreling from Afar: Adapting Squirrel-Net Modules for Remote Teaching and Learning

The shift from face-to-face instruction to remote teaching and learning has proven to be a challenging endeavor for many reasons, including lack of time, resources, and inspiration. Lab courses, the “hands-on” portion of many curricula, may be especially difficult to adapt to online learning given the common use of specialized equipment, materials, and techniques that require close supervision. Without the time and resources to creatively modify existing activities or create new ones, remote lab courses run the risk of becoming less effective, equitable, and/or engaging. Squirrel-Net has created four field-based activities for biology labs that are easy to implement, highly flexible for different course aims, and readily adaptable to a remote learning environment. In this essay, we briefly summarize the modules and propose several ways that each can be adjusted to accommodate online teaching and learning. By providing authentic learning opportunities through distance delivery we hope to promote widespread student engagement and creative solutions for instructors

Sorry to Eat and Run: A Lesson Plan for Testing Trade-off in Squirrel Behavior Using Giving Up Densities (GUDs)

All animals need to find and compete for food, shelter, and mates in order to survive and reproduce. They also need to avoid being eaten by predators. Optimal foraging theory provides a framework to examine the trade-offs individuals make while foraging for food, taking into account an animal’s body condition, predation pressure, quality of food resources, and food patch availability in the habitat. Here we describe an activity that uses Giving Up Densities (GUDs), which could be used as part of a course-based undergraduate research experience (CURE) or as a stand-alone activity. GUDs provide an experimental approach to quantify the costs and benefits of foraging in a particular patch and is simple to measure in that it is literally the density of food remaining in a patch. However, its interpretation allows students to compare foraging decisions under different environmental conditions, between species, or with different food sources. This activity was designed to study the foraging behavior of squirrels, which are active during the day, forage on seeds, and are found on and around many college campuses, but it can be adapted to nocturnal animals, birds, or other vertebrates. This module is hands-on. Students weigh seeds, sift sand, walk out into the field with bags of sand and trays, and analyze data. The module can be designed at various levels of inquiry to suit the needs of a particular class. Further, students can work individually, in pairs, or in teams. Finally, students and instructors are encouraged to upload their data to a national dataset, which is available to instructors for use in the classroom to broaden the possible hypotheses and analyses students can explore

Squirrels in Space: Using Radio Telemetry to Explore the Space Use and Movement of Sciurid Rodents

Biotelemetry is used by researchers to track the interactions of animals with each other and the environment. While advancing technology has led to the development of numerous biotelemetry tools, radio telemetry remains the most common method for tracking small animals. Moreover, telemetry tracking of animal movement is an important skill for entry-level positions in wildlife biology. Thus, hands-on experience using radio telemetry provides students with an advantage as they pursue careers in wildlife biology, as well as an opportunity to build science process skills. We present a lesson in which students use radio telemetry to track animals; collect, analyze and interpret spatial data; and consider its applications to local wildlife management and conservation. Students submit their data to a national database collecting observations from multiple institutions as part of Squirrel-Net (http://squirrel-net.org). The aggregated data allows students to generate and test hypotheses across a broader variety of species and habitats than would be possible at any single institution. The lesson is designed for adaptation to diverse educational contexts, from a single two-hour laboratory period (basic skills acquisition) to a semester-long student-driven research project (open inquiry Course-based Undergraduate Research Experience, or CURE). Although this activity and the national database focus on spatial data for squirrels, which are diurnal, charismatic, easily identified, and present on most college campuses, the same methods and materials can be modified for any animal capable of carrying a radio transmitter and being safely tracked by students

An Introduction to the Squirrel-Net Teaching Modules

Although course-based undergraduate research experiences (CUREs) are gaining popularity in biology, most are designed for benchwork-based laboratory courses while few focus on field-based skills. Many barriers to implementing field CUREs exist, including the difficulty in designing authentic research that can be accomplished in a limited lab timeframe, permitting and liability issues, and problems gathering sufficient data to meaningfully analyze. Squirrel-Net (http://squirrel-net.org) is a consortium of mammalogists from eight different institutions who have worked to overcome these limitations through four field-based CUREs focused on sciurid rodents (e.g., squirrels, chipmunks, marmots, prairie dogs). Each module is linked to a national dataset, allowing for broader and more complex hypotheses and analyses than would be possible from a single institution. Modules have been field tested at different institutions and are easily implemented and highly flexible for different courses, levels of inquiry, habitats, and focal species. Beyond the basic lesson plan, each module also provides suggestions for adaptation at different levels of inquiry and scaffolding across a course or an entire curriculum. Moreover, our website provides templates to help lower barriers to CURE implementation (e.g., selecting a field site and writing institutional animal care protocols). Here, we introduce Squirrel-Net and give an overview of the four CURE modules. Additionally, we demonstrate how the modules can be used singly or together to provide authentic research experiences to a diversity of undergraduates

Squirreling Around for Science: Observing Sciurid Rodents to Investigate Animal Behavior

Hands-on research experiences are important opportunities for students to learn about the nature of inquiry and gain confidence in solving problems. Here, we present an inquiry-based lesson plan that investigates the foraging behavior of sciurid rodents (squirrels) in local habitats. Squirrels are an ideal study system for student research projects because many species are diurnal, easy to watch, and inhabit a range of habitats including college campuses. In this activity, instructors identify appropriate field sites and focal species, while students generate questions and brainstorm predictions in small groups regarding factors that might influence behavioral trade-offs in sciurids. Students conduct observational surveys of local squirrels in pairs using a standardized protocol and upload their data to a national database as part of the multi-institutional Squirrel-Net (http://squirrel-net.org). Instructors access the nationwide dataset through the Squirrel-Net website and provide students with data for independent analysis. Students across the country observe and record a range of squirrel species, including behaviors and habitat characteristics. The national dataset can be used to answer student questions about why squirrels behave in the way they do and for students to learn about authentic analyses regarding behavior trade-offs. Additionally, the lesson is designed to be modified across a range of inquiry levels, from a single two-hour laboratory activity to a unit- or semester-long student-driven course-based research experience. Our activity highlights the value of using observational data to conduct research, makes use of the Squirrel-Net infrastructure for collaboration, and provides students equitable access to field-based projects with small mammals

P2Y2 receptors Transactivate the EGFR/ERB1 and ERB3 Growth Factor Receptors in Human Salivary Gland Cells [abstract]

Abstract only availableThe epidermal growth factor receptor (EGFR/ERB1) plays a key role in the regulation of epithelial cell development, differentiation and in the pathophysiology of hyperproliferative diseases such as cancer. Transactivation of the EGFR/ERB1 by G-protein coupled receptors has been shown to be dependent on proteolytic cleavage of membrane ligands such as heparin binding epidermal growth factor (HBEGF), EGF, transforming growth factor (TGF-), epiregulin, amphiregulin and betacellulin. Utilizing the human submandibular gland (HSG) cell line, we found that activation of the P2Y2 nucleotide receptor (P2Y2R) by its agonist UTP caused a time-dependent activation of EGFR/ERB1; however, neutralizing antibodies to the known ligands to EGFR/ERB1 failed to inhibit the UTP-induced phosphorylation of EGFR/ERB1. EGFR/ERB1 phosphorylation can also be induced by heterodimerization with one of the other ERB family members, ERB2, ERB3, and ERB4. HSG cells express ERB2 and ERB3 but not ERB4. Since ERB2 is a ligandless receptor, ERB3 is the likely dimerizing partner. Our results indicate that P2Y2R activation by UTP phosphorylates ERB3. Heregulin, the only known ligand for ERB3 is expressed in HSGs. Therefore, our results suggest that P2Y2R activation stimulates the formation of ERB3-EGFR/ERB1 heterodimers by cleavage of heregulin and its binding to ERB3