Phantom Limb Pain: Feeling Sensation from a Limb that is No Longer Present and What it Can Reveal About Our Brain Anatomy.

This is a flexible, interrupted video case that uses phantom limb pain as a platform to investigate brain anatomy with a focus on somatosensory cortical mapping and the homunculus. The case begins with a video of neurologist Dr. V.S. Ramachandran interviewing two amputees who experience phantom limb pain (part one). Through Dr. Ramachandran's dialog with amputees, students learn about the paradoxical condition of feeling pain in a limb that does not exist (e.g., phantom limb pain). Students witness Dr. Ramachandran analyzing fMRI data from an amputee, and subsequently learn the somatosensory cortical mapping of the amputee has remarkably changed. Dr. Ramachandran also introduces and demonstrates one form of treatment for phantom limb pain, the mirror box. The video case is supplemented with optional opportunities for further exploration about the mirror box (part two) and somatosensory cortical mapping, via the two-point discrimination test (parts three and four). In part two, students use the primary literature to investigate the effectiveness of the mirror box, and practice skills of interpreting figures. In parts three and four, students conduct a two-point discrimination test (part three) on each other or a person in their residence and analyze class data (part four). Students are led to discover conceptual connections between all four parts of this module. As one example, students are challenged to predict how two-point discrimination data from amputees (interviewed in the video, part one) would compare to students' two-point discrimination data (parts three and four). While the four parts of this learning module are highly interconnected, instructors can choose to selectively implement one or more parts. In addition, each part can be executed in the face-to-face classroom, as out-of-classroom assignment, in a synchronous or non-synchronous video meeting platform, or as a hybrid of these options, providing flexibility for the instructor. This case has been used in a 100-level face-to-face, non-science major course and it has been modified as an online module for a 300 level General Physiology course.

Locate the Lesion: A Project-Based Learning Case that Stimulates Comprehension and Application of Neuroanatomy.

A fictitious patient, Mr. Challenge, is admitted to the emergency room and displays symptoms consistent with damage to the central nervous system. In this problem-based learning case, students are challenged to determine the location of a lesion that is consistent with Mr. Challenge's symptoms. Students discover details about Mr. Challenge's symptoms while exploring three anatomical pathways: corticospinal tract, spinothalamic tract and medial lemniscal pathway. Students make predictions as to which of these pathways may be damaged in Mr. Challenge and defend their predictions based on their research of the function and anatomical location of these tracts. This ultimately leads the student to identifying a single lesion site that can account for Mr. Challenge's symptoms. This case is executed in an undergraduate neuroscience course and would be useful in anatomy and physiology course, as well as other courses that serve students interested in health science related careers.

An Inquiry-Based Approach to Study the Synapse: Student-Driven Experiments Using C. elegans.

Inquiry-based instruction has been well demonstrated to enhance long term retention and to improve application and synthesis of knowledge. Here we describe an inquiry-based teaching module that trains undergraduates as scientists who pose questions, design and execute hypothesis-driven experiments, analyze data and communicate their research findings. Before students design their research projects, they learn and practice several research techniques with the model organism, Caenorhabditis elegans. This nematode is an ideal choice for experimentation in an undergraduate lab due to its powerful genetics, ease and low cost of maintenance, and amenability for undergraduate training. Students are challenged to characterize an instructor-assigned "mystery mutant" C. elegans strain. The "mystery mutant" strain has a defect in cholinergic synaptic transmission. Students are well poised to experimentally test how the mutation impacts synaptic transmission. For example, students design experiments that address questions including: Does the effected gene influence acetylcholine neurotransmitter release? Does it inhibit postsynaptic cholinergic receptors? Students must apply their understanding of the synapse while using their recently acquired research skills (including aldicarb and levamisole assays) to successfully design, execute and analyze their experiments. Students prepare an experimental plan and a timeline for proposed experiments. Undergraduates work collaboratively in pairs and share their research findings in oral and written formats. Modifications to suit instructor-specific goals and courses with limited or no lab time are provided. Students have anonymously reported their surprise regarding how much can be learned from a worm and feelings of satisfaction from conducting research experiments of their own design.

Characterizing Mystery Cell Lines: Student-driven Research Projects in an Undergraduate Neuroscience Laboratory Course.

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.

A conserved neuropeptide system links head and body motor circuits to enable adaptive behavior.

Neuromodulators promote adaptive behaviors that are often complex and involve concerted activity changes across circuits that are often not physically connected. It is not well understood how neuromodulatory systems accomplish these tasks. Here, we show that the Caenorhabditis elegans NLP-12 neuropeptide system shapes responses to food availability by modulating the activity of head and body wall motor neurons through alternate G-protein coupled receptor (GPCR) targets, CKR-1 and CKR-2. We show ckr-2 deletion reduces body bend depth during movement under basal conditions. We demonstrate CKR-1 is a functional NLP-12 receptor and define its expression in the nervous system. In contrast to basal locomotion, biased CKR-1 GPCR stimulation of head motor neurons promotes turning during local searching. Deletion of ckr-1 reduces head neuron activity and diminishes turning while specific ckr-1 overexpression or head neuron activation promote turning. Thus, our studies suggest locomotor responses to changing food availability are regulated through conditional NLP-12 stimulation of head or body wall motor circuits.

Integrins Have Cell-Type-Specific Roles in the Development of Motor Neuron Connectivity.

Formation of the nervous system requires a complex series of events including proper extension and guidance of neuronal axons and dendrites. Here we investigate the requirement for integrins, a class of transmembrane cell adhesion receptors, in regulating these processes across classes of C. elegans motor neurons. We show α integrin/ina-1 is expressed by both GABAergic and cholinergic motor neurons. Despite this, our analysis of hypomorphic ina-1(gm144) mutants indicates preferential involvement of α integrin/ina-1 in GABAergic commissural development, without obvious involvement in cholinergic commissural development. The defects in GABAergic commissures of ina-1(gm144) mutants included both premature termination and guidance errors and were reversed by expression of wild type ina-1 under control of the native ina-1 promoter. Our results also show that α integrin/ina-1 is important for proper outgrowth and guidance of commissures from both embryonic and post-embryonic born GABAergic motor neurons, indicating an ongoing requirement for integrin through two phases of GABAergic neuron development. Our findings provide insights into neuron-specific roles for integrin that would not be predicted based solely upon expression analysis.

Adaptation of sensory neurons to hyalectin and decorin proteoglycans.

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.

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. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Integrin signaling is integral to regeneration.

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.

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

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.