Supported by USDA-NIFA/AFRI 2010-65105-20582, NSF 0918177, and Bedoukian Research Incorporation.

1. Insects
<ol>
<li>Navel orangeworm, Amyelois transitella (Walker) (Lepidoptera:Pyralidae) colony in our laboratory originated from moths collected from almond trees in Fresno, CA. Our insects colony is maintained in environmental chambers (Percival Inc, IA ) at 28 &plusmn; 2&deg;C, 75 &plusmn; 10% relative humidity, and under 16:8h (light:dark) photo regime.</li>
<li>Emerging moths are collected daily, sexed, and transferred to plastic boxes (669 mL lunchbox,13 x 13 cm; height, 4.5 cm, Rubbermaid) provided with up to 10 layers of water soaked paper towels (Thirsty Ultra Absorbent, 27.9 x 27.9 cm; Safeway). Box covers are perforated to allow air circulation. This arrangement provides ~100% RH.</li>
<li>For electrophysiological measurements we use 2-4 days old moths after emergence, which are kept under high humidity.</li>
</ol>
2. Electrophysiological Preparation
<ol>
<li>Moths (males or females) are gently pushed into a pipette tip (200 &mu;L, USA Scientific Inc) that is cut from the top to have a large (ca. 2 mm) tip diameter. A moth is gently pushed through the other end with a humidified tissue paper stub until the antennae and part of the head protrude from the tip.</li>
<li>Exposed head is immobilized by covering the head with a non-drying clay (Claytoon , Van Aken International, CA) leaving a small part of one eye and two antenna exposed. This preparation is placed on a platform of the EAG Micromanipulator MP-12 (Syntech, Germany) between the two electrode holders.</li>
<li>We use glass electrode made from 1.0 mm borosilicate capillary tubing with filament (WPI Inc, FL) and pulled in a Model P-97 micropipette puller (Sutter Instruments, CA) to obtain microelectrodes of less than a micrometer tip. Electrodes are back filled with sensillum lymph ringer (Kaissling and Thorson 1980). One electrode impales the exposed area of the eye under the microscope (Leica MZ75) and serves as reference, while the recording electrode accommodates the two antennae. To facilitate the contact, distal 1-2 antennal segments are cut before being inserted into the recording electrode. For recordings signals from individual sensilla, we slightly modify this setup wherein the recording electrode is impaled under the microscope (Olympus BX51WI; 800x magnification). Action potentials are recorded by inserting the glass electrode at the base of a sensillum. Signals are amplified by a high impedance pre-amplifier (Syntech, Germany) and fed into a UB-IDAC to be analyzed off-line with AUTOSPIKE software (Syntech, Germany). AC signals are band pass filtered between 100 -10,000 Hz and action potentials are extracted by computer using an AUTOSPIKE algorithm that distinguishes their peak-to-trough amplitudes from noise. Responses of individual neurons are calculated as the increase (or decrease) in action potential frequency (spikes per second) over the spontaneous frequency.</li>
</ol>
3. 	Chemical Stimuli and Stimulation Protocol 
<ol>
<li>Chemicals of highest purity are used. A major constituent of the female pheromone blend from the navel orangeworm, (Z,Z)-11,13-hexadecadienal was obtained from Bedoukian Research Inc, CT. Chemicals are diluted, w/v, with glass-distilled hexane to make stock solutions of 10 &mu;g/ &mu;L and decadic dilutions are made. An aliquot (10 &mu;L) of a stimulus chemical dissolved in hexane is loaded onto a Whatman filter paper strip (8x40 mm), the solvent is evaporated by gently shaking for 10 s under a fume hood and the strip is placed in a 5 mL polypropylene syringe (BD Syringes, NJ) from which various volumes are ejected. Hexane alone and an empty syringe serve as control.</li>
<li>The preparation is held in a humidified air stream delivered by the Syntech Stimulus controller (CS-55 model; Syntech, Germany) at 20 mL/sec to which a stimulus pulse of 4 mL/s is added for 500 ms. Signals are recorded for 10 s, starting 2 s before the onset of the stimulus pulse. Antennal preparation is stimulated with 500 ms pulse during which ca. 2 mL of the purified air from a 5 mL polypropylene syringe containing the stimulus is added onto the main air flow. A gap of at least 1 min or more, following high responses, is allowed between stimulations.</li>
</ol>
4. Representative Results
Antennae of restrained moths (Figure 1) stimulated with female pheromone component, (Z,Z)-11,13-hexadecadienal generate robust dose dependent responses (Figure 2). This major pheromone component also elicits dose dependent excitatory responses from long trichodea sensilla (Figure 3A) as displayed in traces of single unit recordings (Figure 3B).
<img src="/files/ftp_upload/2489/2489fig1.jpg" alt="Figure 1" /> 
Figure 1. Live navel orangeworm moth restrained and antenna exposed 
<img src="/files/ftp_upload/2489/2489fig2.jpg" alt="Figure 2" /> 
Figure 2. Dose dependent EAG responses elicited in response to (Z,Z)-11,13-hexadecadienal, a major female pheromone constituent.
<img src="/files/ftp_upload/2489/2489fig3.jpg" alt="Figure 3" /> 
Figure 3. Sensilla trichoid in male navel orangeworm moth respond to (Z,Z)-11,13-hexadecadienal in a dose dependent manner. (A) Antenna of male navel orangeworm moth is multi-segmented and each segment is adorned with a large number of hair like structures, sensilla. A scanning electron micrograph, showing details in the inset (Scale bars are 200 and 50 &mu;M, respectively). (B) Extracellular single-unit recordings from a trichoid sensillum.

<ol>
	<li>Kaissling, K. -. E., Thorson, J., Sattelle, D. B., Hall, L. M., Hildebrand, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+olfactory+sensilla:+structural,+chemical+and+electrical+aspects+of+the+functional+organization.">Insect olfactory sensilla: structural, chemical and electrical aspects of the functional organization.</a> Receptors for Neurotransmitters. Hormones and Pheromones in Insects. , 261-282 (1980).</li></ol>

No conflicts of interest declared.

This novel method developed for restraining a live navel orangeworm moth to measure olfactory signals has proved to be robust and very reliable. We routinely employ this method for isolating and identifying novel attractants from natural host substrates like almonds and pistachios.

<table border="1">
	<tbody>
 	<tr>
 	 <td>Material Name </td>
 	 <td>Catalogue #/Model</td>
 	 <td>Company</td>
 	 <td>Comment</td>
 	</tr>
 	<tr>
 	 <td>Microscope </td>
 	 <td>BX51WI model</td>
 	 <td>Olympus, USA</td>
 	 <td>&nbsp;</td>
 	</tr>
 	<tr>
 	 <td>Stereo microscope</td>
 	 <td>MZ75</td>
 	 <td>Leica Microsystems Inc. USA</td>
 	 <td>&nbsp;</td>
 	</tr>
 	<tr>
 	 <td>1.0 mm borosilicate capillary tubing with filament </td>
 	 <td>1B100F-3</td>
 	 <td>WPI Inc, FL</td>
 	 <td>&nbsp;</td>
 	</tr>
 	<tr>
 	 <td>Micropipette puller</td>
 	 <td>P-97</td>
 	 <td>Sutter Instruments, CA</td>
 	 <td>&nbsp;</td>
 	</tr>
 	<tr>
 	 <td>Stimulus controller </td>
 	 <td>CS-55 model</td>
 	 <td>Syntech, Germany</td>
 	 <td>&nbsp;</td>
 	</tr>
 	<tr>
 	<td>High Impedance pre-amplifiers (Universal Single ended probe)</td>
 	<td>&nbsp;</td>
 	<td>Syntech, Germany</td>
 	<td>&nbsp;</td>
 		</tr>
 	<tr>
 	<td>Amplifier / data-acquisition system (USB-IDAC)</td>
 	<td>&nbsp;</td>
 	<td>Syntech, Germany</td>
 		<td>&nbsp;</td>
		</tr>
 	<tr>
 	<td>EAG Micromanipulator MP-12 </td>
 	<td>&nbsp;</td>
 	<td>Syntech, Germany</td>
 	<td>&nbsp;</td>
 	 	</tr>
 	 	<tr>
 	 <td>(Z,Z)-11,13-hexadecadienal</td>
			<td>&nbsp;</td>
			<td>Bedoukian Research Inc, CT.</td>
 			<td>&nbsp;</td>
	 </tr>
	 <tr>
	 <td>Whatman filter paper</td>
	 <td>1001070</td>
	 <td>Whatman USA</td>
	 <td>&nbsp;</td>
	 </tr>
	 <tr>
	 <td>5 mL polypropylene syringe </td>
	 <td>309633</td>
	 <td>BD Syringes, NJ</td>
	 <td>&nbsp;</td>
	 </tr>
	 <tr>
	 <td>pipette tip (200 &#x03BC;L)</td>
	 <td>1111-0806</td>
	 <td>USA Scientific Inc</td>
	 <td>&nbsp;</td>
	 </tr>
	 <tr>
	 <td>669 mL lunchbox,13 x 13 cm; height, 4.5 cm, </td>
	 <td>&nbsp;</td>
	 <td>Rubbermaid</td>
	 <td>&nbsp;</td>
	 </tr>
	 <tr>
	 <td>Thirsty Ultra Absorbent, 27.9 x 27.9 cm</td>
	 <td>&nbsp;</td>
	 <td>Safeway</td>
	 <td>&nbsp;</td>
	 </tr>
	 <tr>
	 <td>Non-drying clay </td>
	 <td>18150</td>
	 <td>Claytoon , Van Aken International, CA</td>
	 <td>&nbsp;</td>
	 </tr>
	 <tr>
	 <td>Environmental chamber</td>
	 <td>I-30BLL model</td>
	 <td>&nbsp;</td>
	 <td>&nbsp;</td>
	 </tr>
 </tbody>
</table>

electrophysiological measurements from a moth olfactory system

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.

Watch this Scientific Journal Video about Electrophysiological Measurements from a Moth Olfactory System at JoVE.com

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

electrophysiological-measurements-from-a-moth-olfactory-system

Research

JoVE Journal

Neuroscience

13.7K Aufrufe.  University of California, Davis. Insect olfaktorischen Systeme bieten einzigartige Möglichkeiten für die Aufnahme Riechstoff-induzierten Reaktionen in den Formen der Elektroantennogramme (EAG) und Single Sensillum Aufnahmen (SSR), die Antworten zusammengefasst sind von allen Geruchsrezeptor Neuronen (ORNs) an der Antenne befindet und von denen in einzelnen untergebracht Sensillen, jeweils.

Serie: Electrophysiological Measurements from a Moth Olfactory System

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Electrophysiological Measurements and Analysis of Nociception in Human Infants

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.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

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.
Drosophila 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.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

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.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...