Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.
22 Related JoVE Articles!
A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila
Institutions: University of California, Los Angeles.
It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.
Neuroscience, Issue 21, tether, Drosophila, magnet, olfaction, flight, behavior
Vertical T-maze Choice Assay for Arthropod Response to Odorants
Institutions: University of Florida .
Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, various technologies are being developed as control tools. A subset of these tools focuses on modifying the behavior of arthropods by attraction or repulsion. Therefore, arthropods are often the focus of behavioral investigations. Various tools have been developed to measure arthropod behavior, including wind tunnels, flight mills, servospheres, and various types of olfactometers. The purpose of these tools is to measure insect or arachnid response to visual or more often olfactory cues. The vertical T-maze oflactometer described here measures choices performed by insects in response to attractants or repellents. It is a high throughput assay device that takes advantage of the positive phototaxis (attraction to light) and negative geotaxis (tendency to walk or fly upward) exhibited by many arthropods. The olfactometer consists of a 30 cm glass tube that is divided in half with a Teflon strip forming a T-maze. Each half serves as an arm of the olfactometer enabling the test subjects to make a choice between two potential odor fields in assays involving attractants. In assays involving repellents, lack of normal response to known attractants can also be measured as a third variable.
Biochemistry, Issue 72, Molecular Biology, Basic Protocols, Entomology, Behavior, Eukaryota, Organic Chemicals, Chemical Actions and Uses, Life Sciences (General), Behavioral Sciences, Arthropod behavior, chemical ecology, olfactometer, chemotaxis, olfaction, attraction, repulsion, odorant, T-maze, psyllid, Diaphorina citri, insect, anthropod, insect model
Olfactory Assays for Mouse Models of Neurodegenerative Disease
Institutions: University of Cincinnati, University of Cincinnati, Wright State University.
In many neurodegenerative diseases and particularly in Parkinson’s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson’s disease as well as in testing potential disease-modifying therapies.
Neuroscience, Issue 90,
olfaction, mouse, Parkinson’s disease, detection, discrimination, sniffing
Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)
Institutions: Freie Universität Berlin.
Honeybees (Apis mellifera
) are well known for their communication and orientation skills and for their impressive learning capability1,2
. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4
. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12
. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.
Neuroscience, Issue 47, Classical conditioning, behavioural pharmacology, insect, invertebrate, honeybee, learning, memory
Testing Drosophila Olfaction with a Y-maze Assay
Institutions: UMR-6265 CNRS, UMR-1324 INRA, Université de Bourgogne.
Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster
is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.
Neuroscience, Issue 88, environmental effects (biological, animal and plant), genetics (animal and plant), life sciences, animal biology, behavioral sciences, Y-maze, olfaction, adult, choice, behavior, Drosophila melanogaster
Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording
Institutions: Johns Hopkins University.
Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1
, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8
, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13
. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2
. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor.
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.
JoVE Neuroscience, Issue 37, olfaction, electrophysiology, field potential, generator potential, EOG
Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits
Institutions: Washington University in St. Louis .
Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1
. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3
. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5
. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7
. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3
. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8
. We provide details of our experimental setup and present representative recording traces for each of these techniques.
Neuroscience, Issue 71, Neurobiology, Biomedical Engineering, Bioengineering, Physiology, Anatomy, Cellular Biology, Molecular Biology, Entomology, Olfactory Receptor Neurons, Sensory Receptor Cells, Electrophysiology, Olfactory system, extracellular multi-unit recordings, first-order olfactory receptor neurons, second-order projection neurons, third-order Kenyon cells, neurons, sensilla, antenna, locust, Schistocerca Americana, animal model
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana
plants with Agrobacteria
carrying launch vectors. Optimization of Agrobacterium
cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana
, N. excelsiana
× N. excelsior
) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium
harboring pBID4-GFP (Tobacco mosaic virus
-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium
laboratory strain GV3101 showed the highest protein production compared to Agrobacteria
laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria
strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana
resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
Visually Mediated Odor Tracking During Flight in Drosophila
Institutions: University of California, Los Angeles.
Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.
Neuroscience, Issue 23, Drosophila, magnet, olfaction, vision, behavior, flight, video
Perceptual and Category Processing of the Uncanny Valley Hypothesis' Dimension of Human Likeness: Some Methodological Issues
Institutions: University of Zurich.
Mori's Uncanny Valley Hypothesis1,2
proposes that the perception of humanlike characters such as robots and, by extension, avatars (computer-generated characters) can evoke negative or positive affect (valence) depending on the object's degree of visual and behavioral realism along a dimension of human likeness
) (Figure 1
). But studies of affective valence of subjective responses to variously realistic non-human characters have produced inconsistent findings 3, 4, 5, 6
. One of a number of reasons for this is that human likeness is not perceived as the hypothesis assumes. While the DHL can be defined following Mori's description as a smooth linear change in the degree of physical humanlike similarity, subjective perception of objects along the DHL can be understood in terms of the psychological effects of categorical perception (CP) 7
. Further behavioral and neuroimaging investigations of category processing and CP along the DHL and of the potential influence of the dimension's underlying category structure on affective experience are needed. This protocol therefore focuses on the DHL and allows examination of CP. Based on the protocol presented in the video as an example, issues surrounding the methodology in the protocol and the use in "uncanny" research of stimuli drawn from morph continua to represent the DHL are discussed in the article that accompanies the video. The use of neuroimaging and morph stimuli to represent the DHL in order to disentangle brain regions neurally responsive to physical human-like similarity from those responsive to category change and category processing is briefly illustrated.
Behavior, Issue 76, Neuroscience, Neurobiology, Molecular Biology, Psychology, Neuropsychology, uncanny valley, functional magnetic resonance imaging, fMRI, categorical perception, virtual reality, avatar, human likeness, Mori, uncanny valley hypothesis, perception, magnetic resonance imaging, MRI, imaging, clinical techniques
Barnes Maze Testing Strategies with Small and Large Rodent Models
Institutions: University of Missouri, Food and Drug Administration.
Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.
bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.
distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.
random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.
shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.
Behavior, Issue 84, spatial navigation, rats, Peromyscus, mice, intra- and extra-maze cues, learning, memory, latency, search strategy, escape motivation
Appetitive Associative Olfactory Learning in Drosophila Larvae
Institutions: University of Konstanz, University of Fribourg.
In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila
larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative
learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4
. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6
. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila
larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10
larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14
. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9
. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9
Neuroscience, Issue 72, Developmental Biology, Neurobiology, Biochemistry, Molecular Biology, Physiology, Behavior, Drosophila, fruit fly, larvae, instar, olfaction, olfactory system, odor, 1-octanol, OCT, learning, reward, sugar, feeding, animal model
Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees
Institutions: University of Würzburg.
In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1
, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2
connecting the AL with the MB. The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi channel wire electrodes is demonstrated. Pairwise differential amplification of the micro wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long term recordings over many hours up to days, which is a clear advantage compared to conventional extra and intracellular in vivo
Neuroscience, Issue 89, honeybee brain, olfaction, extracellular long term recordings, double recordings, differential wire electrodes, single unit, multi-unit recordings
A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation
Institutions: Faculty of Medicine, Memorial University, University of Victoria.
Rat pups during a critical postnatal period (≤ 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via
single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.
Neuroscience, Issue 90, lateralized odor learning, rats, memory, nose plug, olfactory bulb, piriform cortex, phosphorylated CREB
High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity
Institutions: Monell Chemical Senses Center.
Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11
. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e.
receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors—an essential step toward a model of odor coding in the mammalian olfactory system.
Neuroscience, Issue 88, Firefly luciferase, Renilla Luciferase, Dual-Glo Luciferase Assay, olfaction, Olfactory receptor, Odorant, GPCR, High-throughput
Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches
Institutions: Centre National de la Recherche Scientifique (CNRS), Institut d'Ecologie et des Sciences de l'Environnement de Paris, Institut Pasteur.
Robots designed to track chemical leaks in hazardous industrial facilities1
or explosive traces in landmine fields2
face the same problem as insects foraging for food or searching for mates3
: the olfactory search is constrained by the physics of turbulent transport4
. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6
, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7
but also chemicals that are relevant to humans, e.g.
, volatile compounds emanating from cancer cells8
or toxic and illicit substances9-11
. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ
in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12
. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13
. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14
or using nanostructured gas sensors that mimic insect antennae15
Neuroscience, Issue 90, robotics, electroantennogram, EAG, gas sensor, electronic nose, olfactory search, surge and casting, moth, insect, olfaction, neuron
A Molecular Readout of Long-term Olfactory Adaptation in C. elegans
Institutions: George Washington University, Fred Hutchinson Cancer Research Center, University of California San Francisco .
During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans
was first described by Colbert and Bargmann1,2
. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans
animals are exposed to an attractive AWC-sensed odor3
for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans
animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans
were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4
uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4
. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5
. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7
. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.
Developmental Biology, Issue 70, Neuroscience, Molecular Biology, Cellular Biology, Olfactory adaptation, C. elegans, EGL-4, nuclear translocation, olfaction, animal model
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
A Video Demonstration of Preserved Piloting by Scent Tracking but Impaired Dead Reckoning After Fimbria-Fornix Lesions in the Rat
Institutions: Canadian Centre for Behavioural Neuroscience, University of Lethbridge.
Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; O’Keefe and Nadel, 1978; Mittelstaedt and Mittelstaedt, 1980; Landeau et al., 1984; Etienne, 1987; Gallistel, 1990;
Maurer and Séguinot, 1995). Piloting requires the use of the relationships between relatively stable external (visual, olfactory, auditory) cues, whereas dead reckoning requires the integration of cues generated by self-movement. Animals obtain self-movement information from vestibular receptors, and possibly muscle and joint receptors, and efference copy of commands that generate movement. An animal may also use the flows of visual, auditory, and olfactory stimuli caused by its movements. Using a piloting strategy an animal can use geometrical calculations to determine directions and distances to places in its environment, whereas using an dead reckoning strategy it can integrate cues generated by its previous movements to return to a just left location. Dead reckoning is colloquially called "sense of direction" and "sense of distance."
Although there is considerable evidence that the hippocampus is involved in piloting (O’Keefe and Nadel, 1978; O’Keefe and Speakman, 1987), there is also evidence from behavioral (Whishaw et al., 1997; Whishaw and Maaswinkel, 1998; Maaswinkel and Whishaw, 1999), modeling (Samsonovich and McNaughton, 1997), and electrophysiological (O’Mare et al., 1994; Sharp et al., 1995; Taube and Burton, 1995; Blair and Sharp, 1996; McNaughton et al., 1996; Wiener, 1996; Golob and Taube, 1997) studies that the hippocampal formation is involved in dead reckoning. The relative contribution of the hippocampus to the two forms of navigation is still uncertain, however. Ordinarily, it is difficult to be certain that an animal is using a piloting versus a dead reckoning strategy because animals are very flexible in their use of strategies and cues (Etienne et al., 1996; Dudchenko et al., 1997; Martin et al., 1997; Maaswinkel and Whishaw, 1999). The objective of the present video demonstrations was to solve the problem of cue specification in order to examine the relative contribution of the hippocampus in the use of these strategies. The rats were trained in a new task in which they followed linear or polygon scented trails to obtain a large food pellet hidden on an open field. Because rats have a proclivity to carry the food back to the refuge, accuracy and the cues used to return to the home base were dependent variables (Whishaw and Tomie, 1997). To force an animal to use a a dead reckoning strategy to reach its refuge with the food, the rats were tested when blindfolded or under infrared light, a spectral wavelength in which they cannot see, and in some experiments the scent trail was additionally removed once an animal reached the food. To examine the relative contribution of the hippocampus, fimbria–fornix (FF) lesions, which disrupt information flow in the hippocampal formation (Bland, 1986), impair memory (Gaffan and Gaffan, 1991), and produce spatial deficits (Whishaw and Jarrard, 1995), were used.
Neuroscience, Issue 26, Dead reckoning, fimbria-fornix, hippocampus, odor tracking, path integration, spatial learning, spatial navigation, piloting, rat, Canadian Centre for Behavioural Neuroscience
A Single-fly Assay for Foraging Behavior in Drosophila
Institutions: University of California-San Diego, Columbia University, Dart NeuroScience, University of Pennsylvania.
For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster
. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center.
The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.
Neuroscience, Issue 81, Drosophila, olfaction, neuromodulation, chemotaxis, hunger, nervous system, behavioral sciences
The Resident-intruder Paradigm: A Standardized Test for Aggression, Violence and Social Stress
Institutions: University Groningen, Radboud University Nijmegen.
This video publication explains in detail the experimental protocol of the resident-intruder paradigm in rats. This test is a standardized method to measure offensive aggression and defensive behavior in a semi natural setting. The most important behavioral elements performed by the resident and the intruder are demonstrated in the video and illustrated using artistic drawings. The use of the resident intruder paradigm for acute and chronic social stress experiments is explained as well. Finally, some brief tests and criteria are presented to distinguish aggression from its more violent and pathological forms.
Behavior, Issue 77, Neuroscience, Medicine, Anatomy, Physiology, Genetics, Basic Protocols, Psychology, offensive aggression, defensive behavior, aggressive behavior, pathological, violence, social stress, rat, Wistar rat, animal model
High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae
Institutions: Rockefeller University.
Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.
Neuroscience, issue 11, odor, olfactory, Drosophila, behavior