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 that the dogs are comfortable in lateral recumbency during testing.</li>
	<li>Allow the dog at least 10 min to freely explore and acclimate to the room and become comfortable with the QST operator and handler. Offer fresh water ad libitum in the room, and give occasional food rewards.</li>
	<li>Randomize the testing site (left or right side) by a coin flip. Clip an approximately 2 x 4 cm section of fur centered around the space between the dorsal surface of the third and fourth metatarsals halfway between the tarsometatarsal joint and the metatarsophalangeal joint. Clip an approximately 1 x 2 cm fur section on the lateral antebrachium just proximal to the antebrachiocarpal joint over the ulna.</li>
</ol>
2. Electronic von Frey anesthesiometer
<ol>
	<li>Instrument set-up
	<ol>
		<li>Gently apply a rigid 0.9 mm von Frey tip to the load cell and ensure that the load cell is securely screwed into the handpiece. Connect the cord from the handpiece to the recording device through the M0 channel (Figure 1A,B).</li>
		<li>Turn on the recording device and press the MAX button so that the device will record and display the maximum force achieved when the dog responds to the applied stimulus (Figure 1C).</li>
		<li>Zero the instrument by pressing the CLR button.</li>
	</ol>
	</li>
	<li>Data collection
	<ol>
		<li>Place the dog in lateral recumbency for measuring thresholds. 
		NOTE: Dogs are placed in right lateral recumbency for measuring thresholds on the left limbs or placed in left lateral recumbency for measuring thresholds on the right limbs. If the dog will not willingly lay in lateral recumbency when given verbal cues, the QST operator and handler can manually lay the dog in lateral recumbency.</li>
		<li>Apply minimal to moderate restraint as needed to maintain the dog in lateral recumbency and relatively still. 
		NOTE: The handler performs this step.</li>
		<li>Apply the stimulus once the dog is calm and relaxed and the limb being tested is in at least 70% extension. Provide gentle manual support to the limb being tested to keep the limb off the floor and provide stable backing to apply force against while not preventing the dog from withdrawing the limb. 
		NOTE: The QST operator performs this step.</li>
		<li>Apply the von Frey tip perpendicular to the skin of the area being tested. If the dog exhibits reflex movements (e.g., twitching of the paw or withdrawal of the limb before force is being applied) 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. Take a measurement when the skin does not cause reflex movements by applying the von Frey tip.</li>
		<li>Apply steadily increasing force with the von Frey tip (~20 g/sec) until the dog withdraws the limb, vocalizes, turns to look at the stimulus, or exhibits other movements or 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. 
		NOTE: Do not exceed 1,000 g of force.</li>
		<li>Record the maximum force applied that is displayed on the recording device. 
		NOTE: If the safety cut-off of 1,000 g of force is reached, 1,000 g is recorded as the sensory threshold, and it is noted that there was no response before the safety cut-off.</li>
		<li>Repeat the measurements for a total of five trials, allowing 1 min between each measurement (inter-trial interval). Zero the instrument between each step by pressing the CLR button.
		<ol>
			<li>Allow the dog to remain in lateral recumbency during the inter-trial intervals if they remain relatively calm and relaxed with no or minimal restraint. Otherwise, allow the dog to sit, stand, or move about the QST room to maintain their comfort. Place the dog back in lateral recumbency before the subsequent measurement.</li>
		</ol>
		</li>
		<li>Record a feasibility score of 0-5 to indicate the ease with which the data was collected. 
		NOTE: Feasibility scores are as follows: 0 = no problem, 1 = mild difficulty, 2 = moderate difficulty, 3 = significant difficulty, 4 = extremely difficult, 5 = impossible. The rubric used for assigning feasibility scores is provided in Table 1.</li>
		<li>Give the dog a 5 min break before starting measurements with the blunt probed pressure algometer.</li>
	</ol>
	</li>
</ol>
3. Blunt probed pressure algometer
<ol>
	<li>Instrument set-up
	<ol>
		<li>Ensure that the small blunt probe is securely screwed into the device (Figure 2A).</li>
		<li>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 (g) at the top of the screen (Figure 2B).</li>
		<li>Zero the instrument by pressing the ZERO button.</li>
	</ol>
	</li>
	<li>Data collection
	<ol>
		<li>Place the dog in lateral recumbency for measuring thresholds. 
		NOTE: Dogs are placed in right lateral recumbency for measuring thresholds on the left limbs or placed in left lateral recumbency for measuring thresholds on the right limbs. If the dog will not willingly lay in lateral recumbency when given verbal cues, the QST operator and handler can manually lay the dog in lateral recumbency.</li>
		<li>Apply minimal to moderate restraint as needed to maintain the dog in lateral recumbency and relatively still. 
		NOTE: The handler performs this step.</li>
		<li>Apply the stimulus once the dog is calm and relaxed and the limb being tested is at approximately 70% extension. Provide gentle manual support to the limb being tested to keep the limb off the floor and provide stable backing to apply force against, while not preventing the dog from withdrawing the limb. 
		NOTE: The QST operator performs this step.</li>
		<li>Apply the blunt probe perpendicular to the skin of the area being tested (Figure 2C). If the dog exhibits reflex movements (e.g., twitching of the paw or withdrawal of the limb before force is being applied) from the sensation of the blunt probe on the skin, allow the dog to relax the limb again before reapplying the blunt probe. Take a measurement when the application of the blunt probe to the skin does not cause reflex movements.</li>
		<li>Apply steadily increasing force with the probe (~20 g/s) until the dog withdraws the limb, vocalizes, turns to look at the stimulus, or exhibits other movements or 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. 
		NOTE: Do not exceed 2,500 g of force.</li>
		<li>Record the maximum force applied that is displayed on the recording device. 
		NOTE: If the safety cut-off of 2,500 g of force is reached, 2,500 g is recorded as the sensory threshold, and it is noted that there was no response before the safety cut-off.</li>
		<li>Repeat the measurements for a total of five trials, allowing 1 min between each measure (inter-trial interval). Zero the instrument between each step by pressing the ZERO button.
		<ol>
			<li>Allow the dog to remain in lateral recumbency during the inter-trial interval if they remain relatively calm and relaxed with no or minimal restraint. Otherwise, allow the dog to sit, stand, or move about the QST room to maintain their comfort.&#160;Place the dog back in lateral recumbency before the subsequent measurement.</li>
		</ol>
		</li>
		<li>Record a feasibility score of 0-5 to indicate the ease with which the data was collected. 
		NOTE: Feasibility scores are as follows: 0 = no problem, 1 = mild difficulty, 2 = moderate difficulty, 3 = significant difficulty, 4 = extremely difficult, 5 = impossible.</li>
		<li>Give the dog a 5 min break before starting measurements with the hot thermal probe.</li>
	</ol>
	</li>
</ol>
4. Hot thermal probe
<ol>
	<li>Instrument set-up
	<ol>
		<li>Connect the thermosensory analyzer to the computer via the USB cable and ensure that the 16 x 16 mm thermode is connected to the analyzer. Turn on the analyzer.</li>
		<li>Open the thermosensory analyzer software on the computer and select the TSA II analyzer from the startup menu. Click on OK on the pop-up warning for the analyzer self-test. Ensure that the thermode is not connected to the study subject during the self-test.</li>
		<li>In the TEST tab (upper right-hand corner), under the Select Patient prompt (left-hand side of the screen), select the appropriate patient by double-clicking on the name in the list.
		<ol>
			<li>To create a new patient, click on the PATIENTS tab to the right of the TEST tab. Click on the New Patient icon in the lower left-hand corner and fill in the patient details (department, patient first and last name, ID, gender, and date of birth).</li>
		</ol>
		</li>
		<li>Under the Select Program prompt in the TEST tab, select the appropriate program by double-clicking on the program in the list.
		<ol>
			<li>To create a new program, single-click on the PROGRAMS tab to the right of the PATIENTS tab. Click on the New Program icon in the lower left-hand corner and fill in the program details. 
			NOTE: For this protocol, the program details are given in Table 2. A body site does not need to be selected under the Select Body Site prompt in the TEST tab.</li>
		</ol>
		</li>
		<li>Once the appropriate patient and program have been selected, click on the Go to Test prompt under the TEST tab. Single click on the Pre-Test button in the lower left-hand corner to calibrate the analyzer to the specified program.</li>
		<li>Once the Pre-Test is complete, the Pre-Test button is replaced by the Start button, and the test window displays: Press Start button to start the test (Figure 3A).</li>
		<li>Unwind the thermode cable and ensure that the thermode is easily accessible.</li>
	</ol>
	</li>
	<li>Data collection
	<ol>
		<li>Place the dog in lateral recumbency for measuring thermal latency. 
		NOTE: Dogs are placed in right lateral recumbency for measuring thresholds on the left limbs or placed in left lateral recumbency for measuring thresholds on the right limbs. If the dog will not willingly lay in lateral recumbency when given verbal cues, the QST operator and handler can manually lay the dog in lateral recumbency.</li>
		<li>Apply minimal to moderate restraint as needed to maintain the dog in lateral recumbency and relatively still. 
		NOTE: The handler performs this step.</li>
		<li>Apply the stimulus once the dog is calm and relaxed and the limb being tested is in approximately 70% extension. Provide gentle manual support to the limb being tested to keep the limb off the floor while not preventing the dog from withdrawing the limb. Also, hold and operate a stopwatch with the hand supporting the limb. 
		NOTE: The QST operator performs this step.</li>
		<li>Apply the thermode to the skin of the area being tested (Figure 3B). If the dog exhibits reflex movements (e.g., twitching of the paw or withdrawal of the limb before heat is being applied) from the sensation of the thermode on the skin, allow the dog to relax the limb again before reapplying the thermode. Take a measurement when the application of the thermode to the skin does not cause reflex movements.</li>
		<li>Click on the Start button in the lower left-hand corner of the TEST tab to start the test. 
		NOTE: The QST operator signals to the handler to start the test (e.g., by nodding their head), and the QST operator simultaneously starts the stopwatch.</li>
		<li>Remove the thermode when the dog withdraws the limb, vocalizes, turns to look at the stimulus, or exhibits other movements or behavioral responses that indicate the conscious perception of the stimulus or when the maximum latency is reached while simultaneously stopping the stopwatch. 
		NOTE: The QST operator performs this step. Do not exceed 20 s of thermode application or 49 &#176;C of maximum thermode temperature.</li>
		<li>Record the latency to withdrawal. If the safety cut-off of 20 s of thermode application is reached, record 20 s as the sensory latency, and note that there was no response before the safety cut-off.</li>
		<li>Repeat the measurements for a total of five trials, allowing 1 min between each measure (inter-trial interval). Click on the End Test button, and then the Pre-Test button between each measurement to stop heating the thermode and recalibrate the thermode to prepare for the next application. 
		NOTE: The handler performs this step.
		<ol>
			<li>Allow the dog to remain in lateral recumbency during the inter-trial interval if they remain relatively calm and relaxed with no or minimal restraint. Otherwise, allow the dog to sit, stand, or move about the QST room to maintain their comfort.&#160;Place the dog back in lateral recumbency before the subsequent measurement.</li>
		</ol>
		</li>
		<li>Record a feasibility score of 0-5 to indicate the ease with which the data was collected. 
		NOTE: Feasibility scores are as follows: 0 = no problem, 1 = mild difficulty, 2 = moderate difficulty, 3 = significant difficulty, 4 = extremely difficult, 5 = impossible.</li>
	</ol>
	</li>
</ol>

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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. 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The authors have no conflicts of interest to disclose.

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 ...

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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 ...

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Neuroscience

2.1K Views.  North Carolina State University. 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.

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

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 ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

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.