This research was supported by grants from the Health Research Board, Dystonia Ireland, Science Foundation Ireland and the Irish Institute for Clinical Neuroscience.

The Medical Research Ethics Committee at St. Vincent&#39;s University Hospital, Dublin gave approval for the recruitment of patients with cervical dystonia, their siblings (unaffected by dystonia), and healthy controls, to participate in the protocol described below.

1. Hardware &#38; Software Solutions
Note: Two hardware options have been developed to display visual stimuli with precise inter-stimulus intervals. Both were designed and built in-house at the Trinity Centre for Bioengineering, Trinity College Dublin, and have been previously described5,16. Those wishing to replicate the exact hardware solutions used herein may request same by contacting the Trinity Centre for Bioengineering directly. Alternatively, a full set of instructions including 3D printing files for the headset, instructions for the accompanying Arduino microcontroller, etc. can be downloaded from http://www.dystoniaresearch.ie/temporal-discrimination-threshold/. The stimuli presented in the table top approach may be generated using custom programs in Presentation (e.g., Neurobehavioural Systems), installed on a desktop computer and programmed to control the light-emitting diodes (LED) via the parallel port of the computer. Alternatively, as described below, the table top LEDs may be controlled via an Arduino microcontroller. Both the Presentation code and Arduino files are also available to download from the above link.
<ol>
	<li>TDT hardware: Table-Top Method
	<ol>
		<li>Mark an &#39;X&#39;, as a fixation point, on a black mat or sheet placed on the table in front of the participant.</li>
		<li>Ask the participant to position themselves so that they are sitting directly in front of the fixation point.</li>
		<li>Place the yellow light-emitting diode (LED) pairs (5 mm diameter, 90 cd/m2 luminance), encased in a box, on the table in front of the participant.</li>
		<li>Orient the box such that the LEDs are vertically aligned and positioned 7 &#176; from the subject&#39;s center point on the left and right side, as needed.</li>
		<li>Conduct this experiment in a darkened room. A small amount of background luminance may be required to enable the operator to see enough to run the experiment.</li>
		<li>Instruct the participant to focus on the fixation point at all times and not to look directly at the flashing LEDs.</li>
		<li>Connect the microcontroller to the LED box and follow the on-screen instructions displayed on the liquid crystal display of the microcontroller box, e.g., select presentation method: &#39;random&#39; or &#39;staircase&#39;, and select mode: &#39;left top first&#39;, etc.</li>
		<li>Ask the participant to respond &#34;same&#34; or &#34;different&#34; following presentation of each stimulus pair, depending on whether they perceive the stimuli to be synchronous or asynchronous.</li>
		<li>Inform the participant when each trial is about to commence, by vocalizing the on-screen countdown from 5 - 0 s.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56310/56310fig1.jpg" /> 
Figure 1: (a) Schematic of the design of the headset. A pair of yellow LEDs (5 mm diameter),and the red fixation LED (3 mm diameter), are placed on the left and right side of the participant via a head-mounted unit and made visible by way of reflection in the mirrors in front of the user. (b) Schematic 3D model of the headset. The headset was developed from laser-sintered nylon plastic, weighs 0.70 kg, has a low transparency index and is black in color to minimize light penetrance. (a and b) are reproduced, with slight modification, from Butler et al.16 with permission from IOP Publishing. (c) The LED stimulus box for table-top presentation.
<ol start="2">
	<li>TDT Hardware: Portable TDT Headset
	<ol>
		<li>Conduct the experiment in any suitable location.</li>
		<li>Connect the microcontroller to the headset and follow the on-screen instructions displayed on the liquid crystal display of the microcontroller box, e.g., select presentation method: &#39;random&#39; or &#39;staircase&#39;, and mode: &#39;left top first&#39;, etc.</li>
		<li>Direct the participant to position themselves with their elbows on a table in front of them. Then, holding the device in their hands, direct them to gently press their face into the rubber sealant surrounding the eyepiece, thereby sealing out ambient light.</li>
		<li>Instruct the participant to focus on the red fixation LED at all times and not to look directly at the flashing LEDs.</li>
		<li>Ask the participant to respond &#34;same&#34; or &#34;different&#34; following presentation of each stimulus pair, depending on whether they perceive the stimuli to be synchronous or asynchronous.</li>
		<li>Inform the participant when each trial is about to commence, by vocalizing the on-screen countdown from 5 - 0 s.</li>
	</ol>
	</li>
</ol>
2. Stimulus Presentation
Note: Two approaches to stimulus presentation have been employed.
<ol>
	<li>Staircase method
	<ol>
		<li>Select &#39;staircase&#39; presentation; stimuli are presented every 5 s with the inter-stimulus interval starting at 0 and becoming progressively more asynchronous (increasing by 5 ms) each time.</li>
		<li>Select any of the four presentation modalities: (i) left top LED first (ii) left bottom LED first (iii) right top LED first, or (iv) right bottom LED first.</li>
		<li>Repeat step 2.1.2 so that each modality is run twice, resulting in a total of eight runs.</li>
		<li>Terminate the trial when a participant responds &#34;different&#34; for three consecutive pairs of stimuli.</li>
	</ol>
	</li>
	<li>Random Presentation Method
	<ol>
		<li>Select &#39;Random&#39; presentation; stimuli pairs are presented every 5 s. The inter-stimulus interval varies, in a randomized fashion, from 0-100 ms.</li>
		<li>Select any of the four presentation modalities: (i) left top LED first (ii) left bottom LED first (iii) right top LED first, or (iv) right bottom LED first.</li>
		<li>Repeat step 2.2.2 so that each modality is run twice, resulting in a total of eight runs. 
		Note: Each run is the same length and will complete automatically.</li>
	</ol>
	</li>
</ol>
3. Data Analysis
<ol>
	<li>Single TDT value
	<ol>
		<li>Using the data from the staircase method, highlight the first of the final three &#34;different&#34; responses for each of the eight runs. These are the threshold values for each run.</li>
		<li>Calculate the temporal discrimination threshold (TDT) for each participant by taking the median of the thresholds from each of their eight runs; resulting in a single TDT value (in milliseconds) per individual.</li>
		<li>Calculate the Zscore for each participant. Define the Zscore as the difference between the participant&#39;s TDT, and the mean TDT from an age-matched control population (<img alt="Equaiton 1" src="/files/ftp_upload/56310/56310eq1.jpg" />, divided by the standard deviation of the TDT values for that control population <img alt="Equaiton 2" src="/files/ftp_upload/56310/56310eq2.jpg" />. 
		<img alt="Equaiton 3" src="/files/ftp_upload/56310/56310eq3.jpg" /></li>
		<li>Determine if the individual has a normal or abnormal TDT. A Zscore &#8805; 2.5 is deemed to reflect an abnormal TDT.</li>
	</ol>
	</li>
	<li>Distribution Analysis
	<ol>
		<li>Using the data from the staircase method, encode the response data such that &#39;0&#39; corresponds to &#34;same&#34; and &#39;1&#39; corresponds to &#34;different&#34;, Table 1.</li>
		<li>Download a free MATLAB.exe to perform the distribution analysis described below from http://www.dystoniaresearch.ie/temporal-discrimination-threshold/.&#160;See Butler et al.16 for a full description of this method. Alternatively, proceed as described below.</li>
		<li>Pad out the data to ensure all runs are the same length as the longest run. This is done by assuming all subsequent responses, following termination of a run, are &#34;different&#34;, Table 1(b).</li>
		<li>Average responses across trials for each participant, Table 1(c). This can be plotted as a function of stimulus asynchrony.</li>
		<li>Fit this averaged or representative data with a cumulative Gaussian function. The mean of this distribution represents the point at which participants are equally likely to respond &#8220;same&#8221; or &#8220;different&#8221;. This point is referred to as the &#8216;point of subjective equality&#8217; (PSE). The standard deviation of the Gaussian distribution, also referred to as the &#8216;just noticeable difference&#8217; (JND), indicates how sensitive participants are to changes in temporal asynchrony around their mean.</li>
		<li>Extend the analysis by submitting the data to a non-parametric bootstrapping procedure in order to estimate the 95% confidence intervals for the TDT and the PSE and JND of the psychometric, cumulative Gaussian function. To do this, generate new representative data sets by random sampling with replacement from the original responses, Table 1(b), for each time step. Calculate the TDT and fit a new psychometric function for each representative data set16.</li>
		<li>Calculate the goodness of fit, or deviance (D), for each participant using the log-likelihood ratio,16,17 
		<img alt="Equaiton 4" src="/files/ftp_upload/56310/56310eq4.jpg" /> 
		where K is the number of time points, ni is the number of repetitions at that time point, generally eight repetitions (four right and four left), yi is the observed proportion of asynchronous responses, pi is the proportion of asynchronous responses predicted by the fitted curve. A deviance value of 0 means a perfect fit.</li>
		<li>Plot the results. 
		Note: Data from the random presentation approach can be analyzed to determine the single or distributed TDT as described in section 3 above for data arising from the staircase presentation method. However, due to the random presentation order of inter-stimuli intervals, these data must first be ordered (from smallest to largest inter-stimulus interval), prior to commencing the analysis described above, Table 2. In addition, it is not necessary to pad the data following random presentation as, by default, all runs are of equal length.</li>
	</ol>
	</li>
</ol>

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Rebecca B Beck, Eavan M Mc Govern, John Butler, Dorina Birsanu, Brendan Quinlivan, Ines Beiser, Shruti Narasimham have no funding sources, financial disclosures or conflict of interests to declare. Michael Hutchinson receives research grants from Dystonia Ireland, the Health Research Board of Ireland (CSA-2012-5), Foundation for Dystonia Research (Belgium) and the Irish Institute of Clinical Neuroscience. Sean O&#39;Riordan reports receiving a speaker&#39;s honorarium from Abbvie. Richard Reilly receives funding from Science Foundation Ireland, Enterprise Ireland and the Health Research Board of Ireland.

TDT Measurement and Analysis 
Two forms of apparatus (table-top and headset), two methods of stimulus presentation (staircase and random), and two approaches to data analysis (traditional and distribution) have been presented to illustrate how to measure and quantify a person&#39;s temporal discrimination ability. The portable headset provides a convenient hardware option that ensures consistency in distance and angles between the participant and the LED light sources while al...

Temporal discrimination describes a person&#39;s ability to discriminate, or perceive, rapid changes in their environment. The temporal discrimination threshold (TDT) is the shortest time interval at which an individual can perceive that two sequential sensory stimuli are asynchronous. Temporal discrimination has been shown to be abnormally prolonged in disorders affecting the basal ganglia, including dystonia1,2,3,4,5,6,7.
Dystonia is the third most common neurological movement disorder - after Parkinson&#39;s disease and Essential Tremor. It is characterized by sustained or intermittent muscle contractions causing abnormal, often repetitive, movements or postures8. Dystonia can affect any part of the body. When it affects one body part it is known as focal dystonia8. Dystonia affecting the neck muscles is known as cervical dystonia, and is the most common phenotype of adult onset idiopathic isolated focal dystonia.9,10 The pathogenesis of cervical dystonia remains unknown; it is considered to be a genetic disorder with autosomal dominant inheritance and markedly reduced penetrance. Environmental factors are also considered important in relation to disease penetrance and expression.
The superior colliculus, a sensorimotor structure situated in the dorsal midbrain, is important for the rapid detection of environmental stimuli in the process of covert attentional orienting2,11,12. Visual stimuli access the superior colliculus rapidly through the retino-tectal magnocellular pathway. The TDT is a simple, objective measure believed to represent the processing of visual (and other sensory stimuli) in the superficial layers of the superior colliculus. The TDT has been studied in individuals with cervical dystonia, their unaffected relatives and healthy control participants. Compared to age- and sex-matched control participants, an abnormal TDT has high sensitivity (97%, 36 of 37 patients) and specificity (98-100%) in cervical dystonia1. An abnormal TDT has been found in 50% of unaffected first-degree female relatives of patients with cervical dystonia (14 of 25, aged 48 years or older), demonstrating age- and sex-related penetrance with autosomal dominant inheritance13,14. An abnormal TDT in unaffected relatives of cervical dystonia patients (compared to relatives with normal TDTs) is associated with increased putaminal volume (by voxel-based morphometry)15 and reduced putaminal activity (by fMRI)4. The superior colliculus is considered a significant node in the neuronal network, which is dysfunctional in cervical dystonia12. The assessment of temporal discrimination is regarded as providing important clues as to the pathomechanisms underlying cervical dystonia.
The goal of this article is to present two methods for measuring and analyzing temporal discrimination, as well as demonstrating the application of this method to studying the pathophysiology of cervical dystonia.

<table border="0" cellpadding="0" cellspacing="0" width="1057">
	<tbody>
		<tr height="86">
			<td height="86" style="height:87px;width:216px;">TDT head set</td>
			<td style="width:555px;">Can be supplied by Trinity Centre for Bioengineering, Trinity College Dublin.&#160; Alternatively full instructions are available for free download from http://www.dystoniaresearch.ie/temporal-discrimination-threshold/</td>
			<td style="width:119px;">1</td>
			<td style="width:167px;">A custom-built, portable device for the presentation of visual stimuli.</td>
		</tr>
		<tr height="82">
			<td height="82" style="height:83px;width:216px;">TDT table top LED box</td>
			<td style="width:555px;">Can be supplied by Trinity Centre for Bioengineering, Trinity College Dublin.&#160; Alternatively full instructions are available for free download from http://www.dystoniaresearch.ie/temporal-discrimination-threshold/</td>
			<td style="width:119px;">2</td>
			<td>A custom-built, table-top device for the presentation of visual stimuli.</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:89px;width:216px;">Microcontroller</td>
			<td style="width:555px;">Can be supplied by Trinity Centre for Bioengineering, Trinity College Dublin.&#160; Alternatively full instructions are available for free download from http://www.dystoniaresearch.ie/temporal-discrimination-threshold/</td>
			<td style="width:119px;">3</td>
			<td>A custom-built microcontroller for the delivery of visual stimuli in staircase or random order, with precise inter-stimulus intervals.</td>
		</tr>
	</tbody>
</table>

measurement &amp; analysis of the temporal discrimination threshold applied to cervical dystonia

The temporal discrimination threshold (TDT) is the shortest time interval at which an observer can discriminate two sequential stimuli as being asynchronous (typically 30-50 ms). It has been shown to be abnormal (prolonged) in neurological disorders, including cervical dystonia, a phenotype of adult onset idiopathic isolated focal dystonia. The TDT is a quantitative measure of the ability to perceive rapid changes in the environment and is considered indicative of the behavior of the visual neurons in the superior colliculus, a key node in covert attentional orienting. This article sets out methods for measuring the TDT (including two hardware options and two modes of stimuli presentation). We also explore two approaches of data analysis and TDT calculation. The application of the assessment of temporal discrimination to the understanding of the pathogenesis of cervical dystonia and adult onset idiopathic isolated focal dystonia is also discussed.

Examples of filled score sheets are provided in Tables 1 and 2, where these respectively represent results following staircase and random stimulus presentation methods. The thresholds for each run (the timing of the first of three stimulus pairs deemed to be &#39;different&#39;), are highlighted. In the case of Table 1, the TDT is calculated as 25 ms (i.e., the median of 40, 25, 25, 25, 45, 25, 40, 10 ms). These ...

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Measurement & Analysis of the Temporal Discrimination Threshold Applied to Cervical Dystonia

Methods for the measurement and analysis of the temporal discrimination threshold are presented, and its application to the study of the pathogenesis of cervical dystonia are discussed.

Measurement &amp; Analysis of the Temporal Discrimination Threshold Applied to Cervical Dystonia

The temporal discrimination threshold (TDT) is the shortest time interval at which an observer can discriminate two sequential stimuli as being ...

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9.7K Views.  Trinity College Dublin, The University of Dublin. Methods for the measurement and analysis of the temporal discrimination threshold are presented, and its application to the study of the pathogenesis of cervical dystonia are discussed.

Video: Measurement & Analysis of the Temporal Discrimination Threshold Applied to Cervical Dystonia

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

The Dig Task: A Simple Scent Discrimination Reveals Deficits Following Frontal Brain Damage

Cognitive impairment is the most frequent cause of disability in humans following brain damage, yet the behavioral tasks used to assess cognition in rodent models of brain injury is lacking. Borrowing from the operant literature our laboratory utilized a basic scent discrimination paradigm1-4 in order to assess deficits in frontally-injured rats. Previously we have briefly described the Dig task and demonstrated that rats with frontal brain damage show severe deficits across multiple tests within the task5. Here we present a more detailed protocol for this task. Rats are placed into a chamber and allowed to discriminate between two scented sands, one of which contains a reinforcer. The trial ends after the rat either correctly discriminates (defined as digging in the correct scented sand), incorrectly discriminates, or 30 sec elapses. Rats that correctly discriminate are allowed to recover and consume the reinforcer. Rats that discriminate incorrectly are immediately removed from the chamber. This can continue through a variety of reversals and novel scents. The primary analysis is the accuracy for each scent pairing (cumulative proportion correct for each scent). The general findings from the Dig task suggest that it is a simple experimental preparation that can assess deficits in rats with bilateral frontal cortical damage compared to rats with unilateral parietal damage. The Dig task can also be easily incorporated into an existing cognitive test battery. The use of more tasks such as this one can lead to more accurate testing of frontal function following injury, which may lead to therapeutic options for treatment. All animal use was conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee.

In this protocol, a novel application of scent discrimination is described. The Dig task is a reasonably inexpensive task that can be used to assess frontally-mediated cognition following brain damage.

Cognitive impairment is the most frequent cause of disability in humans following brain damage, yet the behavioral tasks used to assess cognition in ...

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.