Name: assessment of sensory thresholds in dogs using mechanical and hot thermal quantitative sensory testing
Uploaded: 2021-10-26T13:00:00
Description: Watch this Scientific Journal Video about Assessment of Sensory Thresholds in Dogs Using Mechanical and Hot Thermal Quantitative Sensory Testing at JoVE.com

The authors would like to thank Andrea Thomson, Jon Hash, Hope Woods, and Autumn Anthony for handling dogs for QST, Masataka Enomoto for his help screening dogs, and Sam Chiu for his contributions to establishing the protocol for hot thermal QST.

All procedures were approved by the Institutional Animal Care and Use Committee of North Carolina State University.
1. Room set-up and study subject acclimatization
<ol>
	<li>Perform QST in a dedicated space where there is ample room for a QST operator, handler, and dog of any size to move about comfortably. Minimize potential auditory and visual distractions and use a white noise machine to block out ambient sound.</li>
	<li>Place a large yoga mat or similar padding on the floor to ensure t...

<ol>
	<li>Hunt, J., Knazovicky, D., Lascelles, B. D. X., Murrell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+sensory+testing+in+dogs+with+painful+disease:+A+window+to+pain+mechanisms.">Quantitative sensory testing in dogs with painful disease: A window to pain mechanisms.</a> The Veterinary Journal. 243, 33-41 (2019).</li><li>Purves, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+and+subcutaneous+somatic+sensory+receptors.">Cutaneous and subcutaneous somatic sensory receptors.</a> Neuroscience, 2nd edition. , (2001).</li><li>Gorney, A. 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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=von+Frey+anesthesiometry+to+assess+sensory+impairment+after+acute+spinal+cord+injury+caused+by+thoracolumbar+intervertebral+disc+extrusion+in+dogs.">von Frey anesthesiometry to assess sensory impairment after acute spinal cord injury caused by thoracolumbar intervertebral disc extrusion in dogs.</a> The Veterinary Journal. 209, 144-149 (2016).</li><li>Moore, S. A., Hettlich, B. F., Waln, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+an+electronic+von+Frey+device+for+evaluation+of+sensory+threshold+in+neurologically+normal+dogs+and+those+with+acute+spinal+cord+injury.">The use of an electronic von Frey device for evaluation of sensory threshold in neurologically normal dogs and those with acute spinal cord injury.</a> The Veterinary Journal. 197 (2), 216-219 (2013).</li><li>Sanchis-Mora, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pregabalin+for+the+treatment+of+syringomyelia-associated+neuropathic+pain+in+dogs:+A+randomized,+placebo-controlled,+double-masked+clinical+trial.">Pregabalin for the treatment of syringomyelia-associated neuropathic pain in dogs: A randomized, placebo-controlled, double-masked clinical trial.</a> The Veterinary Journal. 250, 55-62 (2019).</li><li>Brydges, N. 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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+thermal+and+mechanical+quantitative+sensory+testing+in+client-owned+dogs+with+chronic+naturally+occurring+pain+and+normal+dogs.">Comparison of thermal and mechanical quantitative sensory testing in client-owned dogs with chronic naturally occurring pain and normal dogs.</a> The Veterinary Journal. 210, 95-97 (2016).</li><li>Knazovicky, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+somatosensory+sensitivity+in+naturally+occurring+canine+model+of+osteoarthritis.">Widespread somatosensory sensitivity in naturally occurring canine model of osteoarthritis.</a> Pain. 157 (6), 1325-1332 (2016).</li><li>Lascelles, B. D. X., Cripps, P. J., Jones, A., Waterman, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-operative+central+hypersensitivity+and+pain:+The+pre-emptive+value+of+pethidine+for+ovariohysterectomy.">Post-operative central hypersensitivity and pain: The pre-emptive value of pethidine for ovariohysterectomy.</a> Pain. 73 (3), 461-471 (1997).</li><li>Slingsby, L. S., Waterman-Pearson, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+post-operative+analgesic+effects+of+ketamine+after+canine+ovariohysterectomy+-+a+comparison+between+pre-+or+post-operative+administration.">The post-operative analgesic effects of ketamine after canine ovariohysterectomy - a comparison between pre- or post-operative administration.</a> Research in Veterinary Medicine. 69 (2), 147-152 (2000).</li><li>Sammarco, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-operative+analgesia+for+stifle+surgery:+A+comparison+of+intra-articular+bupivacaine,+morphine,+or+saline.">Post-operative analgesia for stifle surgery: A comparison of intra-articular bupivacaine, morphine, or saline.</a> Veterinary Surgery. 25 (1), 59-69 (1996).</li><li>Tomas, A., Marcellin-Little, D. J., Roe, S. C., Motsinger-Reif, A., Lascelles, B. D. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+mechanical+thresholds+and+limb+use+in+dogs+with+coxofemoral+joint+OA-associated+pain+and+the+modulating+effects+of+pain+alleviation+from+total+hip+replacement+on+mechanical+thresholds.">Relationship between mechanical thresholds and limb use in dogs with coxofemoral joint OA-associated pain and the modulating effects of pain alleviation from total hip replacement on mechanical thresholds.</a> Veterinary Surgery. 43 (5), 542-548 (2014).</li><li>Briley, J. D., Williams, M. D., Freire, M., Griffith, E. H., Lascelles, B. D. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+and+repeatability+of+cold+and+mechanical+quantitative+sensory+testing+in+normal+dogs.">Feasibility and repeatability of cold and mechanical quantitative sensory testing in normal dogs.</a> The Veterinary Journal. 199 (2), 246-250 (2014).</li><li>Knazovicky, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicate+effects+and+test-retest+reliability+of+quantitative+sensory+threshold+testing+in+dogs+with+and+without+chronic+pain.">Replicate effects and test-retest reliability of quantitative sensory threshold testing in dogs with and without chronic pain.</a> Veterinary Anesthesia and Analgesia. 44 (3), 615-624 (2017).</li><li>Sanchis-Mora, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+initial+validation+of+a+sensory+threshold+examination+protocol+(STEP)+for+phenotyping+canine+pain+syndromes.">Development and initial validation of a sensory threshold examination protocol (STEP) for phenotyping canine pain syndromes.</a> Veterinary Anesthesia and Analgesia. 44 (3), 600-614 (2017).</li><li>Backonja, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Value+of+quantitative+sensory+testing+in+neurological+and+pain+disorders:+NeuPSIG+consensus.">Value of quantitative sensory testing in neurological and pain disorders: NeuPSIG consensus.</a> Pain. 154 (9), 1807-1819 (2013).</li><li>Wylde, V., Palmer, S., Learmonth, I. D., Dieppe, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatosensory+abnormalities+in+knee+OA.">Somatosensory abnormalities in knee OA.</a> Rheumatology. 51 (3), 535-543 (2012).</li></ol>

It is crucial to the acquisition of accurate data - that reflects the dog&#39;s sensory thresholds - that the dog is as calm, relaxed, and positioned adequately as possible for each measurement. A previous study noted that agitation from restraint or distraction from factors within or outside the testing environment affected dogs&#39; responses to the QST stimuli16. If the dog becomes agitated from recumbency or restraint or is distracted, the dog should be given time to settle before a measuremen...

Quantitative sensory testing (QST) assesses the responses elicited by externally applied stimuli; it is used to evaluate the function of the somatosensory system in humans and animals1. Mechanical stimuli in the form of punctate pressure or deep pressure are applied as a ramped stimulus. The sensory threshold is determined as the force that evokes a psychophysical response1. Hot or cold thermal stimuli can be used as a ramped stimulus or as a fixed intensity stimulus. The sensory threshold is determined as the temperature at which there is a response or the latency to respond to the stimulus. Punctate pressure sensory thresholds are measured using electronic von Frey anesthesiometers or von Frey hair filaments, deep pressure is measured using handheld pressure algometers, and thermal sensory thresholds are determined using a variety of contact thermode systems.
QST provides information about the functioning of both peripheral and central sensory pathways and can be used to evaluate alterations in these sensory pathways (algoplasticity) in various disease processes, particularly those that cause chronic pain1. Meissner&#39;s corpuscles detect punctate pressure, and the sensation is transmitted by A&#946; afferent fibers at non-noxious levels and A&#948; afferent fibers when the stimulus is of a noxious intensity1,2. Deep pressure is detected by Pacinian corpuscles and transmitted by C afferent fibers, noxious heat is detected by Ruffini corpuscles and transmitted by A&#948; and C afferent fibers, and noxious cold is detected by Krause corpuscles and transmitted by C afferent fibers1,2. QST can be used to detect both inhibition (decreased sensitivity, hypoesthesia) and facilitation (increased sensitivity, hyperesthesia) of these receptors and pathways. In dogs, QST has been used to evaluate alterations in sensory thresholds secondary to acute spinal cord injury3,4,5, Chiari-like malformation and syringomyelia6, cranial cruciate ligament rupture5,7, and osteoarthritis (OA)8,9,10. Additionally, some studies have used QST to assess pain alleviation provided by certain analgesics6,11,12,13 and surgical procedures14. These studies have provided important insights into the mechanisms of pain sensation in dogs, such as evidence for peripheral and central sensitization after surgery and diseases causing chronic pain states such as cranial cruciate ligament rupture and OA. This information can help improve the detection and treatment of pain in dogs.
Validation studies of mechanical and hot thermal QST in dogs have shown good feasibility, repeatability, and reliability of QST results over time in normal dogs and dogs with chronic pain from OA8,9,15,16. However, several studies have found poor repeatability and reliability of cold thermal and occasionally von Frey QST1,15,17. These studies used different equipment and methodology but provided evidence that mechanical and hot thermal QST is an accurate, semi-quantitative method of measuring sensory thresholds in dogs. However, attention to precise details, including the setting of the measurements, is critical to optimizing QST in dogs, necessitating a standardized protocol for QST. Sanchis-Mora et al. detailed a sensory threshold examination protocol (STEP) for mechanical and hot and cold thermal QST but encountered difficulty with dogs not responding to the cold thermal QST or the highest gram force von Frey filament used in the study17. The following protocol provides a standard method for mechanical and hot thermal QST in dogs; this protocol can assess sensory thresholds in normal dogs or dogs with various disease processes affecting the somatosensory system. The development of standardized protocols may allow for comparing results across studies and meta-analyses of data to improve the utility of QST in veterinary medicine.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Electronic von Frey anesthesiometer</td>
			<td>IITC Life Science Inc.</td>
			<td>Item # 23931</td>
			<td>Custom made with a 1000g max force load cell</td>
		</tr>
		<tr>
			<td>Medoc Main Station software</td>
			<td>Medoc</td>
			<td>(supplied with TSA-II)</td>
			<td></td>
		</tr>
		<tr>
			<td>SMALGO: SMall Animal ALGOmeter</td>
			<td>Bioseb</td>
			<td>Model VETALGO</td>
			<td></td>
		</tr>
		<tr>
			<td>TSA-II NeuroSensory Analyzer</td>
			<td>Medoc</td>
			<td>DC 00072 TSA-II</td>
			<td>No longer manufactured - new model is TSA-2 with same probes and same function</td>
		</tr>
	</tbody>
</table>

assessment of sensory thresholds in dogs using mechanical and hot thermal quantitative sensory testing

Quantitative sensory testing (QST) is used to evaluate the function of the somatosensory system in dogs by assessing the response to applied mechanical and thermal stimuli. QST is used to determine normal dogs&#39; sensory thresholds and evaluate alterations in peripheral and central sensory pathways caused by various disease states, including osteoarthritis, spinal cord injury, and cranial cruciate ligament rupture. Mechanical sensory thresholds are measured by electronic von Frey anesthesiometers and pressure algometers. They are determined as the force at which the dog exhibits a response indicating conscious stimulus perception. Hot thermal sensory thresholds are the latency to respond to a fixed or ramped temperature stimulus applied by a contact thermode.
Following a consistent protocol for performing QST and paying attention to details of the testing environment, procedure, and individual study subjects are critical for obtaining accurate QST results for dogs. Protocols for the standardized collection of QST data in dogs have not been described in detail. QST should be performed in a quiet, distraction-free environment that is comfortable for the dog, the QST operator, and the handler. Ensuring that the dog is calm, relaxed, and properly positioned for each measurement helps produce reliable, consistent responses to the stimuli and makes the testing process more manageable. The QST operator and handler should be familiar and comfortable with handling dogs and interpreting dogs&#39; behavioral responses to potentially painful stimuli to determine the endpoint of testing, reduce stress, and maintain safety during the testing process.

Mechanical and thermal QST has been performed to detect sensory thresholds in both research and client-owned dogs under various clinical conditions, including normal, healthy dogs, dogs with chronically painful conditions such as OA, dogs with acute spinal cord injury, and to assess post-operative pain and effectiveness of analgesics. Though there is a growing body of work on QST in dogs, no normal range of values has been established for any testing modalities. However, several studies have assessed the feasibility and ...

Watch this Scientific Journal Video about Assessment of Sensory Thresholds in Dogs Using Mechanical and Hot Thermal Quantitative Sensory Testing at JoVE.com

Assessment of Sensory Thresholds in Dogs Using Mechanical and Hot Thermal Quantitative Sensory Testing

This work describes a standard protocol for mechanical and hot thermal quantitative sensory testing to evaluate the somatosensory system in dogs. Sensory thresholds are measured using an electronic von Frey anesthesiometer, pressure algometer, and hot contact thermode.

Quantitative sensory testing (QST) is used to evaluate the function of the somatosensory system in dogs by assessing the response to applied mechanical ...

This protocol for assessing sensory thresholds in dogs using quantitative sensory testing or QST will help standardize studies allowing for valuable comparisons across different studies. This technique provides a noninvasive method to measure sensory thresholds in dogs. Our protocol helps guide researchers on best practices to obtain accurate and consistent data.This protocol can provide insight into mechanisms of pain sensation in dogs, including following surgeries and for diseases associated with chronic pain states. The most important aspect of this technique is being cognizant of canine behavior. To ensure safe, accurate testing, all involved must be mindful of the dog's behavior and anxiety levels.Start with arranging the instrument setup by gently applying a rigid 0.9 millimeter Von Frey tip to the load cell, ensuring that the load cell is securely screwed into the handpiece. Then connect the cord from the handpiece to the recording device through the M0 channel. Next, turn on the recording device and press the max button to record and display the maximum force achieved when the dog responds to the applied stimulus.Zero the instrument by pressing the CLR button. To collect the data and measure thresholds, instruct the dog to lay down and then help the dog to the lateral recumbency position. Apply minimal to moderate restraint to maintain the dog in the position and relatively still.Provide gentle manual support to the limb of the dog being tested to keep the limb off the floor and provide a stable backing to apply force against while not preventing the dog from withdrawing the limb. Once the dog is calm and relaxed, with the limb being tested at 70%extension, apply the stimulus once. Place the Von Frey tip perpendicular to the skin of the area being tested.If the dog exhibits reflex movements from the sensation of the Von Frey tip on the skin, allow the dog to relax the limb again before reapplying the Von Frey tip. When the skin does not cause reflex movements, take a measurement by applying the Von Frey tip. Apply steadily increasing force with the Von Frey tip at 20 grams per second until the dog withdraws the limb, vocalizes.turns to look at the stimulus, or exhibits other movements or behavioral responses indicating the conscious perception of the stimulus. Remove the stimulus when the dog withdraws the limb or the maximum force is reached and record the maximum force applied that is displayed on the recording device. Repeat the measurements for five trials, allowing one minute between each measurement with the dog in lateral recumbency if they remain relatively calm and relaxed with no or minimal restraint.Zero the instrument between each step by pressing the CLR button. Next, record a feasibility score of zero to five to indicate the ease of data collection. Give the dog a break of five minutes before starting measurements with the blunt probed pressure algometer.While setting up the instrument, ensure the small blunt probe is securely screwed into the device. Then turn on the recording device and press the max button to continue when prompted on the screen. Press the unit button until the unit is displayed as grams at the top of the screen and push the zero button to zero the instrument.Next, place and prepare the dog in lateral recumbency to measure the thresholds as mentioned before. Apply the blunt probe perpendicular to the skin of the area being tested to apply a steadily increasing force with the probe at 20 grams per second until the dog exhibits behavioral responses that indicate the conscious perception of the stimulus. Remove the stimulus when the dog withdraws the limb or the maximum force is reached.After recording the maximum applied force from the recording device, repeat the measurements for a total of five trials. Allow the dog to remain in lateral recumbency to sit, stand, or move about the QST room to maintain comfort during the inter-trial interval. Then replace the dog in lateral recumbency before the subsequent measurement.Record a feasibility score of zero to five to indicate the ease with which the data was collected. Give the dog a break of five minutes before starting measurements with the hot thermal probe. Before starting the experiment, prepare the dog as explained before.Then apply the thermode to the skin of the area being tested and click on the start button in the lower left-hand corner of the test tab to start the test. When the dog exhibits movements or behavioral responses that indicate the conscious perception of the stimulus or when the maximum latency is reached, remove the thermode while simultaneously stopping the stopwatch. Record the latency to withdraw.Repeat the measurements for a total of five trials by first clicking the stop button and then clicking the pre-test button in between each measurement to stop heating the thermode. This will allow the software to recalibrate the thermode to prepare for the next application. Record a feasibility score of zero to five to indicate the ease with which the data was collected.The representative analysis summarizes the mechanical and hot thermal QST data from a group of healthy pain-free dogs. The data from the electronic Von Frey aesthesiometer sensory threshold measurements displayed no significant effect of body weights on the sensory thresholds. A similar impact of the body weights was noticed in the data from blunt probed pressure algometer sensory thresholds.On the other hand, the data collected for the hot thermal probe sensory latency showed a significant effect of the body weight on the sensory latency. The overall average, standard deviation, and range of sensory thresholds for the mechanical and hot thermal QST were summarized in the same group of healthy pain-free dogs. This provides reference values for future researchers using these techniques It is most important to reliably identify the dog's behavioral response that indicates the conscious perception of the stimulus which may look different for different dogs.In addition to quantitative sensory testing, collecting behavioral information through validated owner questionnaires and emotional reactivity tests can help provide a comprehensive assessment of the dog's affective state.

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Neuroscience

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities

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.

We describe a protocol to examine the development of opioid-induced hyperalgesia and tolerance in mice. Based on the measurement of thermal and mechanical nociceptive responses of na&#239;ve and morphine-treated animals, it allows to quantify the increase in pain sensitivity (hyperalgesia) and decrease in analgesia (tolerance) associated with chronic opiate administration.

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain ...

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...