isolation-culture-dissociated-sensory-neurons-from-chick

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Assumption College

Aggrecan is a chondroitin sulfate (CS)/keratan sulfate (KS)-substituted proteoglycan (PG) abundant in cartilage which is also present within the mammalian embryonic, adult, and injured adult central nervous system (CNS). Although its role within the CNS is not clear, cell culture studies show that when substituted with CS, aggrecan inhibits neurite extension. To better understand the inhibitory effect of aggrecan on injured adult axons in vivo, we developed a model to independently test intact aggrecan and CS-depleted aggrecan core glycoprotein. Acute rat spinal cord hemisection cavities were filled with a growth-promoting matrix, Matrigel, and severed dorsal rootlets were placed into this matrix. This created an assay in which axons readily grew. The extent of ingrowth in this baseline assay was compared to the ingrowth in Matrigel loaded with intact aggrecan or the purified core glycoprotein of aggrecan. Our results show that both intact aggrecan and equivalent concentrations of the core glycoprotein component significantly inhibit axonal growth in this model system. These results confirm that aggrecan can inhibit the growth of adult axons in vivo and suggest that the inhibitory effects of aggrecan may be mediated, at least in part, by structures located on the core glycoprotein in the absence of the bulk of the CS chains.

Intact aggrecan and chondroitin sulfate-depleted aggrecan core glycoprotein inhibit axon growth in the adult rat spinal cord.

Proteoglycans are abundantly expressed in the pathways of developing and regenerating neurons, yet the responses of neurons to specific proteoglycans are not well characterized. We have shown previously that one chondroitin sulfate proteoglycan (CSPG), aggrecan, is potently inhibitory to sensory axon extension in short-term assays and that over time, embryonic neurons adapt to aggrecan-mediated inhibition through the transcriptional upregulation of integrin expression (Condic et al., 1999). Here, we have compared the response of embryonic sensory neurons to structurally distinct CSPGs that belong to either the hyalectin (or lectican) family of large, aggregating proteoglycans or the decorin (or small leucine-rich proteoglycan) family of smaller proteoglycans. Both of these structurally diverse proteoglycan families are expressed in developing embryos and inhibit outgrowth of embryonic sensory neurons in short-term cultures. These results document a previously uncharacterized inhibitory function for the decorin-family proteoglycan biglycan. Interestingly, embryonic neurons adapt to these diverse proteoglycans over time. Adaptation is associated with upregulation of select integrin alpha subunits in a proteoglycan-specific manner. Overexpression of specific integrin alpha subunits improves neuronal regeneration on some but not all decorin-family CSPGs, suggesting that neurons adapt to inhibition mediated by closely related proteoglycans using distinct mechanisms. Our findings indicate that CSPGs with diverse core proteins and distinct numbers of chondroitin sulfate substitution sites mediate a similar response in sensory neurons, suggesting that increased integrin expression may be an effective means of promoting axonal regeneration in the presence of diverse inhibitory proteoglycan species in vivo.

Adaptation of sensory neurons to hyalectin and decorin proteoglycans.

The inability of the adult injured mammalian spinal cord to successfully regenerate is not well understood. Studies suggest that both extrinsic and intrinsic factors contribute to regeneration failure. In this review, we focus on intrinsic factors that impact regeneration, in particular integrin receptors and their downstream signaling pathways. We discuss studies that address the impact of integrins and integrin signaling pathways on growth cone guidance and motility and how lessons learned from these studies apply to spinal cord regeneration in vivo.

Integrin signaling is integral to regeneration.

Inquiry-based projects promote discovery and retention of key concepts, increase student engagement, and stimulate interest in research. Described here are a series of lab exercises within an undergraduate upper level neuroscience course that train students to design, execute and analyze their own hypothesis-driven research project. Prior to developing their own projects, students learn several research techniques including aseptic cell culture, cell line maintenance, immunocytochemistry and fluorescent microscopy. Working in groups, students choose how to use these techniques to characterize and identify a "mystery" cell line. Each lab group is given a unique cell line with either a neural, astrocyte, or Schwann cell origin. Working together, students plan and execute experiments to determine the cellular origin and other unique characteristics of their mystery cell line. Students generate testable hypotheses, design interpretable experiments, generate and analyze data, and report their findings in both oral and written formats. Students receive instructor and peer feedback throughout the entire project. In summary, these labs train students the process of scientific research. This series of lab exercises received very strong positive feedback from the students. Reflections on student feedback and plans for future improvements are discussed.

Michele L. Lemons

Department of Natural Sciences

Assumption College

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos