In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.
18 Related JoVE Articles!
Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities
Institutions: Université de Strasbourg.
Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols.
We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.
Neuroscience, Issue 89, mice, nociception, tail immersion test, tail pressure test, morphine, analgesia, opioid-induced hyperalgesia, tolerance
A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)
Institutions: University of New Hampshire, Massachusetts Division of Marine Fisheries, Boston University, Middle College.
Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female's seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George's Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.
Environmental Sciences, Issue 84, sperm limitation, spermatophore, lobster fishery, sex ratios, sperm receptacle, mating, American lobster, Homarus americanus
Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis
Institutions: Harvard University.
The amphipod Parhyale hawaiensis
is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale
has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster
. In contrast to the syncytial cleavages of Drosophila
, the early cleavages of Parhyale
are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale
embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale
embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale
cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.
Developmental Biology, Issue 85, Amphipod, experimental embryology, micromere, germ line, ablation, developmental potential, vasa
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise
Institutions: University of Kentucky, Brock University.
The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings with a force transducer to monitor the strength of contraction. In addition, we demonstrate how to visually monitor peristaltic activity, which can be used as a bioassay for various peptides, biogenic amines and neurotransmitters. This preparation is amenable to student laboratories in physiology and for demonstrating pharmacological concepts to students. This preparation has been in use for over 100 years, and it still offers much as a model for investigating the generation and regulation of peristaltic rhythms and for describing the mechanisms underlying their modulation. The pharmacological assays and receptor sub-typing that were started over 50 years ago on the hindgut still contribute to research today. This robust preparation is well suited to training students in physiology and pharmacology.
Neuroscience, Issue 47, invertebrate, crayfish, student laboratory, physiology
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
A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats
Institutions: Concordia University.
Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.
Behavior, Issue 91, Behavioral neuroscience, alcoholism, relapse, addiction, Pavlovian conditioning, ethanol, reinstatement, discrimination, conditioned approach
Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice
Institutions: Duquesne University.
Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.
Medicine, Issue 86, Bladder pain, electromyogram (EMG), interstitial cystitis/bladder pain syndrome (IC/BPS), urinary bladder distension (UBD), visceromotor response (VMR)
Measures of Heart and Ventilatory Rates in Freely Moving Crayfish
Institutions: University of Kentucky.
The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an impending predator attack. Although it is not known whether invertebrates posses an autonomic nervous system identical to that of vertebrates, evidence shows invertebrates have a sympathetic-like response to regulate the internal environment and ready the organism to act behaviorally to a given stimuli. Furthermore, this physiological response can be feasibly measured and it acts as a biological index for the animal's internal state. Measurements of the physiological response can be directly related to internal and external stressors through changes in the central nervous system controlled coordination of the cardio-vascular and respiratory systems. More specifically, monitoring heart and ventilation rates provide quantifiable measures of the stress response not always behaviorally observed. Crayfish are good model organisms for heart and ventilatory rate measurements due to the feasibility of recording, as well as the rich history known of the morphology of the crayfish, dating back to Huxley in 1888, and the well-studied typical behaviors.
Physiology, Issue 32, invertebrate, autonomic nervous system, behavior, crustacean
Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats
Institutions: Wright State University, Universidade São Judas Tadeu.
Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e.
, increased sensitivity to noxious stimulus, and allodynia i.e.,
hypersensitivity to normally innocuous stimuli1
. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2
. However, as stated by Bölcskei et al
, determination of "pain
" in animal models is challenging due to its subjective nature3
. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3
. For instance, by using the hot-plate method of Ankier4
, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 °C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5
. As the recently described method of Bölcskei et al.6
, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Almási et al 5
and Bölcskei et al 3
. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.
Neuroscience, Issue 63, Diabetes, painful diabetic neuropathy, nociception, thermal nociceptive threshold, nocifensive behavior
Use of the Operant Orofacial Pain Assessment Device (OPAD) to Measure Changes in Nociceptive Behavior
Institutions: University of Florida College of Dentistry, University of Florida College of Medicine , Stoelting Co., University of Florida .
We present an operant system for the detection of pain in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) assesses pain behaviors in a more clinically relevant way by not relying on reflex-based measures of nociception. Food fasted, hairless (or shaved) rodents are placed into a Plexiglas chamber which has two Peltier-based thermodes that can be programmed to any temperature between 7 °C and 60 °C. The rodent is trained to make contact with these in order to access a reward bottle. During a session, a number of behavioral pain outcomes are automatically recorded and saved. These measures include the number of reward bottle activations (licks) and facial contact stimuli (face contacts), but custom measures like the lick/face ratio (total number of licks per session/total number of contacts) can also be created. The stimulus temperature can be set to a single temperature or multiple temperatures within a session. The OPAD is a high-throughput, easy to use operant assay which will lead to better translation of pain research in the future as it includes cortical input instead of relying on spinal reflex-based nociceptive assays.
Behavior, Issue 76, Neuroscience, Neurobiology, Anatomy, Physiology, Medicine, Biomedical Engineering, Surgery, Neurologic Manifestations, Pain, Chronic Pain, Nociceptive Pain, Acute Pain, Pain Perception, Operant, mouse, rat, analgesia, nociception, thermal, hyperalgesia, animal model
Meal Duration as a Measure of Orofacial Nociceptive Responses in Rodents
Institutions: Texas A&M University Baylor College of Dentistry.
A lengthening in meal duration can be used to measure an increase in orofacial mechanical hyperalgesia having similarities to the guarding behavior of humans with orofacial pain. To measure meal duration unrestrained rats are continuously kept in sound attenuated, computerized feeding modules for days to weeks to record feeding behavior. These sound-attenuated chambers are equipped with chow pellet dispensers. The dispenser has a pellet trough with a photobeam placed at the bottom of the trough and when a rodent removes a pellet from the feeder trough this beam is no longer blocked, signaling the computer to drop another pellet. The computer records the date and time when the pellets were taken from the trough and from this data the experimenter can calculate the meal parameters. When calculating meal parameters a meal was defined based on previous work and was set at 10 min (in other words when the animal does not eat for 10 min that would be the end of the animal's meal) also the minimum meal size was set at 3 pellets. The meal duration, meal number, food intake, meal size and inter-meal interval can then be calculated by the software for any time period that the operator desires. Of the feeding parameters that can be calculated meal duration has been shown to be a continuous noninvasive biological marker of orofacial nociception in male rats and mice and female rats. Meal duration measurements are quantitative, require no training or animal manipulation, require cortical participation, and do not compete with other experimentally induced behaviors. These factors distinguish this assay from other operant or reflex methods for recording orofacial nociception.
Behavior, Issue 83, Pain, rat, nociception, myofacial, orofacial, tooth, temporomandibular joint (TMJ)
Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats
Institutions: University of New South Wales .
Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2
. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3
). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2
), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4
. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al
(1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6
. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3
). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.
Medicine, Issue 61, Neuropathic pain, sciatic nerve, chronic constriction injury, pain hypersensitivity
Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus
Institutions: Macalester College.
Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen, or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.
Neuroscience, Issue 82, Natural Science Disciplines, Life Sciences (General), Behavioral Sciences, mechanical hyperalgesia, mice, electronic pressure meter, inflammation, snake venom
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
Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays
Institutions: BioSentinel Inc., Madison, WI.
Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay's use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro
replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT's proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.
Neuroscience, Issue 85, Botulinum, food testing, detection, quantification, complex matrices, BoTest Matrix, Clostridium, potency testing
Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards
Institutions: Macquarie University.
Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.
Neuroscience, Issue 2, Visual sensitivity, motion perception, operant conditioning, speed, coherence, Jacky dragon (Amphibolurus muricatus)
Electrophysiological Measurements and Analysis of Nociception in Human Infants
Institutions: University College London, Great Ormond Street Hospital, University College Hospital, University of Oxford.
Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.
Neuroscience, Issue 58, pain, infant, electrophysiology, human development