The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.
22 Related JoVE Articles!
The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern
Institutions: University of Cologne.
Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of abdominal ganglia (A1 to A6). This chain of ganglia includes the part of the central nervous system (CNS) that drives coordinated locomotion of the pleopods (swimmerets): the swimmeret system. It is known for over five decades that in crayfish each swimmeret is driven by its own independent pattern generating kernel that generates rhythmic alternating activity 1-3
. The motor neurons innervating the musculature of each swimmeret comprise two anatomically and functionally distinct populations 4
. One is responsible for the retraction (power stroke, PS) of the swimmeret. The other drives the protraction (return stroke, RS) of the swimmeret. Motor neurons of the swimmeret system are able to produce spontaneously a fictive motor pattern, which is identical to the pattern recorded in vivo 1
The aim of this report is to introduce an interesting and convenient model system for studying rhythm generating networks and coordination of independent microcircuits for students’ practical laboratory courses. The protocol provided includes step-by-step instructions for the dissection of the crayfish’s abdominal nerve cord, pinning of the isolated chain of ganglia, desheathing the ganglia and recording the swimmerets fictive motor pattern extracellularly from the isolated nervous system.
Additionally, we can monitor the activity of swimmeret neurons recorded intracellularly from dendrites. Here we also describe briefly these techniques and provide some examples. Furthermore, the morphology of swimmeret neurons can be assessed using various staining techniques. Here we provide examples of intracellular (by iontophoresis) dye filled neurons and backfills of pools of swimmeret motor neurons. In our lab we use this preparation to study basic functions of fictive locomotion, the effect of sensory feedback on the activity of the CNS, and coordination between microcircuits on a cellular level.
Neurobiology, Issue 93, crustacean, dissection, extracellular recording, fictive locomotion, motor neurons, locomotion
Dorsal Column Steerability with Dual Parallel Leads using Dedicated Power Sources: A Computational Model
In spinal cord stimulation (SCS), concordance of stimulation-induced paresthesia over painful body regions is a necessary condition for therapeutic efficacy. Since patient pain patterns can be unique, a common stimulation configuration is the placement of two leads in parallel in the dorsal epidural space. This construct provides flexibility in steering stimulation current mediolaterally over the dorsal column to achieve better pain-paresthesia overlap. Using a mathematical model with an accurate fiber diameter distribution, we studied the ability of dual parallel leads to steer stimulation between adjacent contacts on dual parallel leads using (1) a single source system, and (2) a multi-source system, with a dedicated current source for each contact. The volume conductor model of a low-thoracic spinal cord with epidurally-positioned dual parallel (2 mm separation) percutaneous leads was first created, and the electric field was calculated using ANSYS, a finite element modeling tool. The activating function for 10 um fibers was computed as the second difference of the extracellular potential along the nodes of Ranvier on the nerve fibers in the dorsal column. The volume of activation (VOA) and the central point of the VOA were computed using a predetermined threshold of the activating function. The model compared the field steering results with single source versus dedicated power source systems on dual 8-contact stimulation leads. The model predicted that the multi-source system can target more central points of stimulation on the dorsal column than a single source system (100 vs. 3) and the mean steering step for mediolateral steering is 0.02 mm for multi-source systems vs 1 mm for single source systems, a 50-fold improvement. The ability to center stimulation regions in the dorsal column with high resolution may allow for better optimization of paresthesia-pain overlap in patients.
Medicine, Issue 48, spinal cord stimulation, dorsal columns, current steering, field steering
Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey
Institutions: University of Edinburgh, Temple University School of Medicine, Temple University School of Medicine.
After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16
°C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 °C. For in situ
hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.
Neuroscience, Issue 92, spinal cord injury, axonal guidance molecules, neurofilaments, regeneration
DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord
Institutions: Max Delbrück Center for Molecular Medicine.
Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1
. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3
. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4
), however, other options have been discussed as well5
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.
Neuroscience, Issue 58, neurons, axonal branching, DRG, Spinal cord, DiI labeling, cGMP signaling
Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats
Institutions: School of Allied Medical Professions, The Ohio State University, Drexel University College of Medicine.
Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8
. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3
. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5
. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5
. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5
. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.
Medicine, Issue 62, Rat, neuropathic pain, allodynia, tactile sensation, spinal cord injury, SCI, von Frey monofilaments
Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro
Institutions: University of Central Florida.
The development of more predictive and biologically relevant in vitro
assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro,
leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.
Bioengineering, Issue 92, cantilever, in vitro, contraction, skeletal muscle, NMJ, cardiomyocytes, functional
Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI
Institutions: University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario.
Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.
Neuroscience, Issue 84, Central Nervous System, Ear, Animal Experimentation, Models, Animal, Functional Neuroimaging, Brain Mapping, Nervous System, Sense Organs, auditory cortex, BOLD signal change, hemodynamic response, hearing, acoustic stimuli
A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity
Institutions: Université de Versailles Saint-Quentin-en-Yvelines, Hôpital Ambroise Paré, Université de Versailles Saint-Quentin-en-Yvelines.
A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.
Physiology, Issue 87, rat, cervical spinal cord injury, respiratory deficit, crossed phrenic phenomenon, respiratory neuroplasticity
Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises
Institutions: University of Kentucky, University of Toronto.
This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.
Basic Protocols, Issue 47, Invertebrate, Crayfish, Modeling, Student laboratory, Nerve cord
Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises
Institutions: University of Kentucky, University of Kentucky, University of Oregon.
The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.
Neuroscience, Issue 80, Crustacean, joint, Muscle, sensory, teaching, educational, neuroscience
Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo
Institutions: Washington University in St. Louis , Nagoya University, Washington University in St. Louis .
The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo
electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1
iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6
. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7
, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo
are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11
. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12
. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13
. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14
. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.
Neuroscience, Issue 76, Neurobiology, Physiology, Cellular Biology, Molecular Biology, Fluorescent imaging, weakly electric fish, sensory processing, tract-tracing, electrophysiology, neuron, individual axons, labeling, injection, surgery, recording, mormyrids, animal model
Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System
Institutions: Ohio University.
Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1
, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2
(Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3
. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5
. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8
. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9
, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8
. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.
Medicine, Issue 59, neuroscience, muscle, electromyography, physiology, TMS, strength, motor control. sarcopenia, dynapenia, lumbar
Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises
Institutions: University of Kentucky, University of Toronto.
The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+
on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.
Neuroscience, Issue 47, Invertebrate, Crayfish, neurophysiology, muscle, anatomy, electrophysiology
Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals
Institutions: University of Illinois at Chicago.
Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+
into the presynaptic terminal, through voltage-gated calcium channels (VGCCs) located in the release face membrane, is the trigger for vesicle fusion and neurotransmitter release. Crucial to the rapidity of synaptic transmission is the spatial and temporal synchrony between the arrival of the action potential, VGCCs and the neurotransmitter release machinery. The ability to directly record Ca2+
currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca2+
and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca2+
entry has been characterized using imaging techniques and macroscopic current measurements – techniques that do not have sufficient temporal resolution to visualize Ca2+
entry. The characterization of VGCCs directly at single presynaptic terminals has not been possible in central synapses and has thus far been successfully achieved only in the calyx-type synapse of the chick ciliary ganglion and in rat calyces. We have successfully addressed this problem in the giant reticulospinal synapse of the lamprey spinal cord by developing an acutely dissociated preparation of the spinal cord that yields isolated reticulospinal axons with functional presynaptic terminals devoid of postsynaptic structures. We can fluorescently label and identify individual presynaptic terminals and target them for recording. Using this preparation, we have characterized VGCCs directly at the release face of individual presynaptic terminals using immunohistochemistry and electrophysiology approaches. Ca2+
currents have been recorded directly at the release face membrane of individual presynaptic terminals, the first such recording to be carried out at central synapses.
Neuroscience, Issue 92, reticulospinal synapse, reticulospinal axons, presynaptic terminal, presynaptic calcium, voltage-gated calcium channels, vesicle fusion, synaptic transmission, neurotransmitter release, spinal cord, lamprey, synaptic vesicles, acute dissociation
Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo
Institutions: Jena University Hospital, Jena, Germany, Jena University Hospital, Jena, Germany.
Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo
in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo
recording has several advantages compared to ex vivo
isolated spinal cord preparations, e.g.
the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.
Neuroscience, Issue 85, Central Nervous System Diseases, Spinal Cord Diseases, Electrophysiology, dorsal root potentials (DRP), spinal cord, GABA, presynaptic inhibition, primary afferent depolarization (PAD), in vivo electrophysiology
Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord
Institutions: Drexel University College of Medicine.
Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of communication with the brain. One of the goals of our research is to promote axon regeneration to restore connectivity across the lesion site. To accomplish this we developed a peripheral nerve (PN) grafting technique where segments of sciatic nerve are either placed directly between the damaged ends of the spinal cord or are used to form a bridge across the lesion. There are several advantages to this approach compared to transplantation of other neural tissues; regenerating axons can be directed towards a specific target area, the number and source of regenerating axons is easily determined by tracing techniques, the graft can be used for electrophysiological experiments to measure functional recovery associated with axons in the graft, and it is possible to use an autologous nerve to reduce the possibility of graft rejection. In our lab we have performed both autologous (donor and recipient are the same animal) and heterologous (donor and recipient are different animals) grafts with comparable results. This approach has been used successfully in both acute and chronic injury situations. Regenerated axons that reach the distal end of the PN graft often fail to extend back into the spinal cord, so we use microinjections of chondroitinase to degrade inhibitory molecules associated with the scar tissue surrounding the area of SCI. At the same time we have found that providing exogenous growth and trophic molecules encourages longer distance axonal regrowth into the spinal cord. Several months after transplantation we perform a variety of anatomical, behavioral and electrophysiological tests to evaluate the recovery of function in our spinal cord injured animals. This experimental approach has been used successfully in several spinal cord injury models, at different levels of injury and in different species (mouse, rat and cat). Importantly, the peripheral nerve grafting approach is effective in promoting regeneration by acute and chronically injured neurons.
Neurobiology, Issue 33, transplantation, SCI, regeneration, tract tracing, electrophysiology
Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo
Institutions: Northwestern University Feinberg School of Medicine.
The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo
, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo
therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1
): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e.
the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro
preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4
, including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo
in adult mice5,6
. However, these recordings were obtained in paralyzed animals, i.e.
without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9
, or reproducting human disease10,11
, we expect this technique to be an essential tool for the study of spinal motor system.
Neuroscience, Issue 70, Physiology, Biophysics, Anatomy, Medicine, Motor System, Spinal Cord, Intracellular Recordings, Motoneurons, EMG, Force, lumbar, neuron, brain, mouse, animal model
An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time
Institutions: University of Louisville, University of Calgary.
Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo
living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo
spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo
spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g.,
calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo
); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.
Neuroscience, Issue 93, spinal cord injury, axon, myelin, two-photon excitation microscopy, Nile Red, axonal degeneration, axonal dieback, axonal retraction
Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica
Institutions: Case Western Reserve University , Case Western Reserve University , Case Western Reserve University .
In animals with large identified neurons (e.g.
mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4
. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5
. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5
, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia
) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7
. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g.
in the suspended buccal mass preparation8
or in vivo9
. This process can also be applied in other motor pools10,11,12
or in other animal systems2,3,13,14
Neuroscience, Issue 73, Physiology, Biomedical Engineering, Anatomy, Behavior, Neurobiology, Animal, Neurosciences, Neurophysiology, Electrophysiology, Aplysia, Aplysia californica, California sea slug, invertebrate, feeding, buccal mass, ganglia, motor neurons, neurons, extracellular stimulation and recordings, extracellular electrodes, animal model
Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception
Institutions: University of Kentucky.
The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive information for an animal. An additional objective of this experiment is to learn anatomy of the preparation by staining, dissection and viewing of neurons and sensory structures under a dissecting microscope. This is performed by using basic neurophysiological equipment to record the electrical activity from a joint receptor organ and staining techniques. The muscle receptor organ (MRO) system in the crayfish is analogous to the intrafusal muscle spindle in mammals, which aids in serving as a comparative model that is more readily accessible for electrophysiological recordings. In addition, these are identifiable sensory neurons among preparations. The preparation is viable in a minimal saline for hours which is amenable for student laboratory exercises. The MRO is also susceptible to neuromodulation which encourages intriguing questions in the sites of modulatory action and integration of dynamic signals of movements and static position along with a gain that can be changed in the system.
Neuroscience, Issue 45, invertebrate, sensory, crayfish, Student Laboratory
Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes
Institutions: National Institute of Neurological Disorders and Stroke, National Institutes of Health.
Retrograde labeling of neurons is a standard anatomical method1,2
that has also been used to load calcium and voltage-sensitive dyes into neurons3-6
. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.
Neuroscience, Issue 62, Retrograde labeling, Fluorescent dyes, Spinal cord, Nerves, Spinal tracts, Optical imaging, Electrophysiology, Calcium-sensitive dyes
Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish
Institutions: Oregon Health and Sciences University.
Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo
preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo
imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.
Neuroscience, Issue 45, Zebrafish, synapse, electrophysiology, patch clamp, acetylcholine receptor, neuromuscular, cholinergic/action potential, myasthenic syndrome, motor control