Name: denver papillae protocol for objective analysis of fungiform papillae
Uploaded: 2015-06-08T14:00:00
Duration: 10 min 50 s
Description: Watch this Scientific Journal Video about Denver Papillae Protocol for Objective Analysis of Fungiform Papillae at JoVE.com

This work was funded in part by Science Education Partnership Award (SEPA) R25 RR025066 between 2008-2012 from the National Institutes of Health and the National Center for Research Resources. Thank you to colleagues in the taste field for thoughtful discussion of the literature, our results and feedback on the use of DPP in various laboratory scenarios: John Hayes, Sue Coldwell, Paul Breslin, Carla Schubert. Thank you also to leadership at the Denver Museum of Nature &#38; Science and the staff in support of citizen science within the Genetics of Taste Lab within Expedition Health. Finally, thank you to all of our volunteer citizen scientists for their time and dedication to developing and using this protocol, including: Bear Aragon, Mike Archer, Su Ataman, Michael Bagley, A&#8217;nette Bertrand, Diana Boyles, Wendy Covert, Sasha Dooley, Patty Drever, Jessica Ern, Laura Harmacek, Sean Hibbard, Joyce Hutchens, Matt Joo, Leta Keane, Willem Leenhouts, Stephania Lukjan, Ashley Matthews, Mike Mauser, Stephanie Miller, Hallie Morgan-Rodriguez, Griffin Scherma, Alyssa Schickedanz, Terri Simon, Dylan Thomas, Rudy Torres, Tyler Wilson, Diane Woltkamp.

The images scored in this study were collected as part of a larger taste study conducted in the Genetics of Taste Lab at the Denver Museum of Nature &#38; Science (the Museum)10. Subjects of the larger study were museum visitors (n = 1195) ranging in age from 18-93. While subjects came from six of the seven continents, participants were primarily of European descent. Each subject&#8217;s data was collected in one 30 min&#160;lab session. The Western Institutional Review Board approved the protocol, informed written consent was given, and subjects volunteered their time and were not compensated for their participation in the study.
1. Data Collection
<ol>
	<li>Image Capture
	<ol>
		<li>Show subjects a photograph of how to pose themselves (Figure 1) and what the captured image will look like. 
		<img alt="Figure 1" src="/files/ftp_upload/52860/52860fig1.jpg" />Figure 1: Image Capture Pose. This is the photograph shown to subjects in the GoT Lab in order to correctly pose for their tongue photo.</li>
		<li>Direct subjects to dry their tongue with a paper hand towel and leave tongue protruding from their mouths.</li>
		<li>Apply approximately 3 ml&#160;of blue food dye at a 1:36 concentration to the apex of the tongue using a sterile, rayon applicator with a 1-inch tip. Have subjects return tongue to their mouth and swallow to remove excess dye.</li>
		<li>Have subjects pose themselves as shown in Figure 1, with their chin on their hands and elbows propped on a table for stability. Direct subjects to extend their tongue a comfortable distance and secure it gently between their teeth.</li>
		<li>Adhere a 2.5 cm piece of filter paper with a 10 mm diameter circular cutout punched in it to the tip of the anterior of the left side of the tongue next to midline (see Figure 1).</li>
		<li>Take at least three close-up images of the tongue, capturing the entire 10 mm circular cutout to ensure visualization of all FP within that area. Use the macro setting of a high-quality digital point and shoot camera attached to a tripod for stability.
		<ol>
			<li>Zoom in to the extent that the entire cutout is photographed while still within the camera&#8217;s recommended zoom range. Ensure that the plane of the camera lens is parallel to the plane of the tongue and that the cutout appears circular in the photo instead of oblong. Review photos to ensure they are high quality and useful for counting.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Image Selection and Preparation
	<ol>
		<li>Upload the photos to the computer.</li>
		<li>Of the images taken of the same subject&#8217;s tongue, select only one for further analysis based on clear definition between structures on the tongue at the highest zoom and the least distorted angle so the tongue is broad and flat toward the camera.</li>
		<li>Open the selected raw image in the open source software, ImageJ. Click on &#8220;Plugins&#8221; and in the dropdown menu scroll to &#8220;Analyze&#8221; and click &#8220;Cell Counter.&#8221; When the cell counter opens, click &#8220;Initialize&#8221; to link the image to the cell counter. 
		Note: Here, use ImageJ 1.45 sec with 32-bit Java 1.6.0_10 version.
		<ol>
			<li>Use a standard magnification of 50% for consistency between scorers and for use in subsequent steps to score FP. Move this image to the left side of the screen next to the cell counter. This image will be referred to as Copy A (See Figure 2, left side of screen).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Open a second copy of the raw image (copy B) for measuring the diameters of the papillae being quantified. Zoom in and out as needed to the preference of the individual scorer throughout the scoring process. Move this copy to the right side of the screen so the two copies are not accidentally confused. (See Figure 2, right side of screen)</li>
	<li>Click on the line tool. Using copy B, zoom in as much as necessary to accurately draw a diameter across the 10 mm inner circle of the filter paper at any angle and click &#8220;Analyze&#8221; and &#8220;Set Scale.&#8221; Fill in the &#8220;Known Distance&#8221; to be &#8220;10.&#8221; Verify the scale is correct by measuring an alternate angle and ensure the diameter range is between 9.8-10.2 mm. If it is not, repeat this step.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/52860/52860fig2.jpg" /> 
Figure 2: Copy A and Copy B. Copy A (left) is linked to the cell counter window and remains at 50% magnification for consistency from scorer to scorer while copy B (right) may be zoomed to the preference of the individual.
2. Scoring FP Using the Denver Papillae Protocol Dichotomous Key
<ol>
	<li>For each candidate papilla, use the following dichotomous key (step 2.2-2.5) detailing the criteria to determine if it is a FP. Refer to Figure 3 for a visual of papillae that are classified as FP and for ones that are rejected at each step in the process. 
	<img alt="Figure 3" src="/files/ftp_upload/52860/52860fig3.jpg" />Figure 3: FP vs. Rejected Papillae. Figure 3a is a tongue with several qualifying FP while 3b-3e have circled areas that violate each rule. (3b) area is amorphous; (3c) papillae are too small; 3d) papilla is blue compared to those surrounding it, (3e) papilla is recessed compared to those around it.</li>
	<li>Shape
	<ol>
		<li>Determine if the candidate papilla is amorphous (shapeless). At 50% zoom see if there is a commonly-recognized geometric shape (oval, cuboidal, round).</li>
		<li>If there is a geometric shape (see Figure 3a), move to step 2.3.</li>
		<li>If there is no geometric shape (see Figure 3b), go to the cell counter window and click &#8220;Type 1.&#8221; On copy A (50% zoom), click the candidate papilla to mark it as amorphous and not a FP. Clicking on types 1-4 in this and subsequent steps allows scorers to know they have addressed all candidate papillae and categorizes which rule was violated when rejecting a structure which will aid in the discussion in step 2.7.2.</li>
	</ol>
	</li>
	<li>Color
	<ol>
		<li>Refer to copy A at 50% zoom and determine if there is any color differentiation over the surface of the tongue. If so, continue to the next 2.3.2. If not, do not use color as a determining factor; move to step 2.4.</li>
		<li>Determine if the candidate papilla is lighter than the tissue or papillae surrounding it (see Figure 3a). If any part of the candidate papilla has remained pink or stained lighter, move to step 2.4.</li>
		<li>If the candidate papilla is blue and the surrounding papillae are lighter (see Figure 3c), go to the cell counter window and click &#8220;Type 2&#8221; on the cell counter. On copy A, click the candidate to mark as too blue and not a FP.</li>
	</ol>
	</li>
	<li>Size
	<ol>
		<li>Using copy B, zoom in to the distance where one can comfortably see the outline of the candidate papilla.</li>
		<li>Click on the line tool and measure across the longest dimension of the candidate papilla. Click &#8220;Analyze&#8221; and then &#8220;measure.&#8221; If the measured length is 0.5 mm or greater (see Figure 3a), move to step 2.5.</li>
		<li>If the measured length is 0.499 mm or less, measure once more to ensure accuracy. If it is still 0.499 mm or less (see Figure 3d), click &#8220;Type 3&#8221; on the cell counter window and on copy A, click the candidate papilla to mark it as too small and not a FP.</li>
	</ol>
	</li>
	<li>Recession
	<ol>
		<li>Using copy A, assess if the candidate papilla is either uniform height with the rest of the tongue or elevated. If the papilla is in a crevice, determine its height compared to the other structures in the crevice, not the surface of the tongue. If the papilla is lower than the papillae surrounding it (see Figure 3e), use &#8220;Type 4&#8221; and on copy A, click the candidate papilla to mark it as recessed and not a FP.</li>
		<li>If the candidate papilla is either of uniform height with the rest of the tongue or elevated (see Figure 3a), use &#8220;Type 5&#8221; to click it and mark that this is a FP. This type 5 total is the raw FP score. Do not close the copies at this point as ImageJ does not save the scores on the cell counter nor the colored marks from types 1-5 on copy A.</li>
	</ol>
	</li>
	<li>Saving Copy A
	<ol>
		<li>Write down in notebook raw FP score.</li>
		<li>On the cell counter window, click &#8220;Export Image.&#8221; Save with the desired naming system of the lab. This will allow the opening of copy A in the future to retain the colored marks from types 1-5. It will not save the raw FP score.</li>
	</ol>
	</li>
	<li>Quality Control
	<ol>
		<li>Score each photo by two individual scorers and compare7.</li>
		<li>If the higher FP raw score is within 10% of the lower FP raw score, average the two scores together for a consensus FP score. If the two FP raw scores differ by more than 10%, pull both scored photos onto the screen.
		<ol>
			<li>Use counter types 1-4 to aid in discussion of discrepancies and confer on a final consensus score. If no consensus can be reached during the discussion, take a break from scoring. At a later point, rescore the image and discuss. If a consensus still cannot be reached, pull in a third scorer. Ask them to score the image and dialogue with the other two.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Training Scorers to Use DPP
<ol>
	<li>Background on Training
	<ol>
		<li>Have two scorers count a variety of images, scoring individually and conferring regularly to ensure that they were using the same criteria. Only when individual counts were consistently within 10% of each other for each the images when scored was the method (finalized above) considered acceptable and termed DPP.</li>
		<li>Choose 15 official images and 15 training images (Supplemental Figures 2-17) in sequential order to score. If an image was deemed uncountable during selection, skip the image and choose next sequential image. 
		Note: These images vary in quality and size as they were chosen while the Lab was still mastering the correct photography technique. One of the selected images was deemed uncountable through this primary counting process but was left in to ensure that scorers were able to make that call when needed. Therefore there are 16 images given to trainees though only 15 scored images.</li>
		<li>Using DPP, have the above-mentioned scorers count the 15 training images and create a consensus score for each.</li>
		<li>Give trainees the 15 official images and ask to score using Miller &#38; Reedy to create a baseline. Record scores. Conduct a group training which followed step 3.2. Once step 3.2 was completed, have the scorers score the 15 official images again and record their scores.</li>
	</ol>
	</li>
	<li>Training Subsequent Scorers on DPP
	<ol>
		<li>Give training scorers the first training image and demonstrate DPP for a few papillae on this image. Discuss reasoning for selecting or rejecting each papilla as an FP. Allow trainees to continue practicing scoring for the rest of that photo and confer with the consensus number (for the GoT Lab&#8217;s consensus number and Type 1-5 markers, see supplementary Figure 1). For this photo discuss any discrepancies regardless of the 10% to ensure understanding of DPP.</li>
		<li>Give subsequent scorers the second training image and have them score using DPP.</li>
		<li>If the trainee raw FP score is within ten percent of the established consensus count from section 3.1.3., allow trainee to move onto the next image and repeat steps.</li>
		<li>If the score is outside the ten percent range, use the trainee&#8217;s colored marks representing types 1-5 to understand and identify the inconsistency and then address discrepant understanding of DPP.</li>
		<li>Give the trainee the opportunity to rescore the image and verify the new score once more. If successful, trainee moves onto the next image until all images are completed.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Using DPP, the variance within image counts across independent scorers and within-scorer&#8217;s counts decreased significantly. While this method makes visual FP density analysis without videomicroscopy less subjective, it should be noted that this method only scores FP. DPP cannot guarantee that structures classified as FP have taste pores and are therefore gustatory papillae nor that the counts are reflective of real taste bud density20,23.The nature of this method is that it establishes reliability through consistency and precision; however, it may be biasing results in a consistent direction. To test accuracy relative to other methods using a true measure of taste bud density (e.g., videomicroscopy) would extend beyond the scope of this study.However, by characterizing the morphology of FP more stringently and creating a dichotomous key, this method addresses Miller &#38; Reedy&#8217;s charge to the field that more characterization of FP needs to be done in order to have consensus on their morphology and quantification2. Additionally, they indicated that this characterization might &#8220;clarify some of the differences among subjects and explain some of the disparate results among investigators2.&#8221; By providing a uniform method of quantification, many of the discrepancies on the role of FP in taste and nutrition-related research may be rectified.
The dichotomous key may be adjusted for the different image capture techniques. The known distance for the measurement scale can easily be set to the diameter used in each lab and recession can be discounted if saran wrap or glass were placed over the tongue for image capture. In the GoT Lab, it was found that the neutral tongue provided the best definition of papillae. This may be due to the amount of light in the Lab which caused saran wrap or glass to reflect light back into the camera creating a glare that made assessment difficult. Some labs have said they find the dye more challenging to count, but the GoT Lab found a diluted blue food dye at a concentration of 1:36 provided the ideal contrast between fungiform and filiform papillae on the majority of tongues.
The creation of a dichotomous key for FP characterization will allow for researchers in different labs, using a variety of image capture methods, to consistently analyze structures on the tongue and have confidence in the reporting of their results. Finally, use of a dichotomous key may have purposes in other areas of scientific methodology where qualities of structures or objects are providing inconsistent numbers from study to study.

Papillae are the visible bumps on the tongue&#8217;s surface. There are four types of papillae: fungiform (FP), foliate, circumvallate, and filiform. While filiform papillae are spread over the surface of the tongue, circumvallate are localized only on the back of the tongue, and foliate are found on the sides. In contrast, FP are located toward the front of the tongue. FP, foliate, and circumvallate papillae are considered gustatory because they have the potential to contain taste buds while filiform do not. Taste buds are clusters of cells responsible for detecting chemical tastants and transducing the stimuli into a gustatory signal in the brain. Saliva and food molecules enter into the taste buds through openings called taste pores where taste molecules then have the potential of activating taste cells.
Interestingly, there are large individual differences in taste bud density, as first observed in a histological study of tongues from 18 cadavers (Miller, 1988)1. Taste buds themselves are not visible on the tongue&#39;s surface, however, taste pores are visible when using proper magnification, lighting, and staining techniques which led Miller &#38; Reedy (1990) to develop a method to identify taste pores and fungiform papillae using videomicroscopy in living human beings2. A number of labs have modified their original videomicroscopy technique, using still photography and little or no magnification. However, since fungiform papillae are much easier to visualize than are pores using these latter techniques, labs turned to counting fungiform papillae in place of taste pores. In doing so, it is assumed that fungiform papilla density is a reasonable proxy measure for taste pore density, even though many fungiform papillae do not contain taste pores or taste buds3; not only are FP more visible and accessible than circumvallate and foliate papillae, but their image capture is less time consuming than taste pore analysis through the alternative method of videomicroscopy2,4. In 1990, Miller &#38; Reedy used this technique to find that taste pore densities are correlated with FP densities3 which was further corroborated in Bartoshuk et al.5 In addition to this anatomical relationship, Miller &#38; Reedy also found that the 8 individuals in the upper half of the taste pore density distribution also rated sucrose, NaCl, and propylthiouracil (PROP) as significantly more intense than did the other 8 individuals3. Subsequent work by Bartoshuk et al. (1994) observed a correlation between suprathreshold PROP taste intensity and fungiform papilla density, as well as taste pore density, in 42 subjects5. This convenient method led to influential studies reporting that subjects whose tongues have many papillae elicit a stronger reaction to many tastants, including the bitter tastants phenylthiocarbamide (PTC) and PROP 6,7.
PTC and PROP are well-researched tastants oft used due to the clear link between a person&#8217;s taste sensitivity phenotype and their genotype. Population studies have shown that people who have the homozygous recessive diplotype of the gene, TAS2R38, have a significantly lower sensitivity to PROP than carriers of the dominant or heterozygous variant of the gene8-10. This heritable phenomenon was first reported in 1932 when Arthur L. Fox announced his discovery of &#8220;taste blindness&#8221; to PTC11. Among people able to taste these bitter compounds, the intensity of taste reported can vary anywhere from slightly unpleasant to excruciatingly bitter6. To account for variance that cannot be explained by TAS2R38, the theory was put forth that it could be attributed to the density of FP on the tongue4.
As taste research progressed, the field was divided into two schools of thought around this theory concerning the role of FP and bitter taste sensitivity. Although many studies corroborated the original claim that FP density is a factor in PROP sensitivities4,6,12,13, there has been a small, but important contingency that has found evidence to refute it, reporting an inability to replicate the role10,14,15,16. Delwiche et al. (2001) warned that the trend is highly variable; FP density does not uniformly account for differences in bitterness sensitivity across subjects, and was only demonstrable for subjects with at least a moderate sensitivity to PROP7. In addition to the use of the less rigorously defined Miller &#38; Reedy method on how to quantify FP, it is important to note that with exception of the Beaver Dam Offspring Study15 conducted by Fischer et al., most of the above studies had small sample sizes which may also contribute to inconsistent findings on the role of FP in taste.
Briefly, in Miller &#38; Reedy&#8217;s seminal methodology paper (1990), the authors stained tongues blue to quantify FP because FP remain pink while filiform papillae absorb the dye and turn blue. They classified FP as &#8220;rounded pink structures about 0.5 mm in diameter&#8221;2. While the authors advised that before using this method for further analytical work, FP characteristics should be more strictly defined and concluded that the variation already noted in the literature &#8220;might be attributable to different subject populations, different methods, and different investigators&#8221;2, their research has led to the commonly accepted characteristics that FP are round17,18, large2, pink or stained lighter2, and elevated4. Taken at face value, these characteristics appear very straight forward. In the Genetics of Taste Lab (GoT Lab or Lab), the use of the method was complicated by the scorers&#8217; individual attempts at qualifying and prioritizing the importance of the above characteristics when some, but not all, of the characteristics were present. These complications included small papillae that appeared pink in the midst of much larger papillae; the entire tongue absorbing the blue dye thus eliminating classification based on color variation; oblong papillae instead of round; and tongues having no variation in elevation, all of which contributed to scorers reporting widely-varied FP counts of the same image.
These variations in analysis led us to review the literature in order to pinpoint the subjectivity, identify any exceptions to the commonly accepted FP characteristics and ultimately devise a protocol to accurately and objectively define and score FP.
First, FP shape is described in the literature as a rounded, mushroom-like structure17,18. However, notable exceptions were also mentioned. Miller emphasized that the term &#8220;fungiform&#8221; is a misnomer and that many FP are not mushroom-shaped but can vary widely in size and morphology18. Melis et al. (2013) also described several papillae that were considered &#8220;distorted,&#8221; where the FP diameter in one direction was at least two standard deviations longer than in another direction19. Furthermore, Cheng &#38; Robinson found that when indicating FP by staining, they ranged from flat-topped to elongated in appearance20. Noting the above exceptions, the characterization of an FP being round and mushroom-shaped appears too narrow a definition.
Second, FP color is described as &#8220;pink circles against a blue background&#8221;7 after staining the tongue with blue dye. Though staining lighter was the most consistent criterion of FP throughout the literature it was still not a uniform qualifier. Cheng &#38; Robinson observed that FP were not always lightly stained, making identification difficult and uncertain20. It appears then that tongues absorb dye differently and while the contrast is apparent on many tongues, some become entirely blue with no distinction while others immediately lose any trace of blue dye21.
The third characteristic, FP size, was fairly consistent among papers, ranging from 0.5 mm to 0.97 mm6,18. In the literature Miller noted that there were two size ranges of papillae on the dorsal anterior of the tongue. The papillae with larger diameters were primarily fungiform and conical papillae, while the papillae with a smaller diameter were mostly filiform18. However, with the exception of the Beaver Dam Offspring Study21, size did not appear to determine if a structure is a FP in the literature, but was provided for papillae already classified as &#8220;fungiform&#34;.
Finally, elevation is listed as the fourth FP characteristic4 though Miller pointed out that distinction between filiform and fungiform papillae is difficult when using this criteria near the margin of the tongue. He gave two examples of FP that varied greatly in elevation, one being 0.8 mm in height while another was less than 0.1 mm in height, but still had two taste pores that were clearly observed18. This observation was also followed up and confirmed by Shahbake et al.4
These definitions and their reported exceptions show that the taste field has long recognized the inconsistency in characterizing FP and the shortcomings of the commonly-used protocol from Miller &#38; Reedy. Indeed, Miller &#38; Reedy exhorted the field to further define the features of FP2. We hypothesized that scorers were giving different weight to the characteristics and prioritizing their importance differently when papillae failed to meet every criterion, such as when a papilla was large and mushroom shaped, but stained blue. It was reasoned that investigators of the previously published studies likely did the same which, when combined with small sample sizes, may account for the varied results coming from use of this method.
The GoT Lab addressed this gap in FP methodology by developing a guideline that defines each characteristic using objective measurements and prioritizes the characteristics to indicate which takes precedence in analysis. This method is the Denver Papillae Protocol (DPP), a dichotomous key with clear and distinct characteristics of FP to ensure accurate and repeatable counts from digital photographs of the tongue. DPP is a proposed method to standardize the previously used Miller &#38; Reedy method, thus removing individual interpretation and ensuring more consistent results when analyzing FP density. By doing so, DPP can be used to more confidently determine the role of FP in taste.
The tongue images taken of the subjects were analyzed by the citizen scientists (scorers) of the Lab who are the true subjects of this study. The Lab&#8217;s core of citizen scientists is comprised of members from the community ranging in age (years) from 16 to mid-80s. A summative evaluation report commissioned by the Museum in 2012 describes citizen scientists as having a strong interest in science and with two-thirds having earned a college degree in a scientific discipline22. Candidate citizen scientists are currently recruited via word of mouth, must successfully complete an interview process with veteran volunteers at the Museum, and undergo a trial period in the Museum&#8217;s permanent health exhibition, Expedition Health, before they have the opportunity to apply for a position in the Lab. Once accepted into the Lab, citizen scientists undergo a 12-week training to become certified to enroll human subjects. Following this certification, they are able to take training modules on various techniques in the Lab (e.g. papillae counting, DNA extraction) and following additional certifications and regular quality control measures, these citizen scientists have the opportunity to actively participate in data analysis. Citizen scientists volunteer their time and are not compensated for their contributions to the Museum.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Image J</td>
			<td>National Institutes of Health</td>
			<td><a href="http://rsb.info.nih.gov/ij">http://rsb.info.nih.gov/ij</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Food dye, Deep Blue Shade</td>
			<td>Esco</td>
			<td>http://escofoods.com/blue-food-coloring.html</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Rayon Tipped OB/GYN Applicators</td>
			<td>Puritan</td>
			<td>25-808 1PR</td>
			<td></td>
		</tr>
		<tr>
			<td>Grade 1 Filter Paper</td>
			<td>Whatman</td>
			<td>Whatman No.: 1001-325</td>
			<td></td>
		</tr>
		<tr>
			<td>Coolpix P100 (10.3 megapixels) and P500 (12.1 megapixels)</td>
			<td>Nikon</td>
			<td>http://imaging.nikon.com/lineup/coolpix/</td>
			<td>neither model appears to be available on the website any longer</td>
		</tr>
		<tr>
			<td>Paper Hand Towels</td>
			<td>Kleenex</td>
			<td>https://www.kleenex.com/HandTowelsDetail.aspx</td>
			<td></td>
		</tr>
	</tbody>
</table>

denver papillae protocol for objective analysis of fungiform papillae

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure consistent scoring between scorers. This protocol builds off of a need that has arisen from the last two decades of taste research using FP as a proxy for taste pore density. FP density has historically been analyzed using Miller &#38; Reedy&#8217;s 1990 characterizations of their morphology: round, stained lighter, large, and elevated. In this work, the authors forewarned that stricter definitions of FP morphology needed to be outlined. Despite this call to action, follow up literature has been scarce, with most studies continuing to cite Miller &#38; Reedy&#8217;s original work. Consequently, FP density reports have been highly variable and, combined with small sample sizes, may contribute to the discrepant conclusions on the role of FP in taste sensitivity. The Genetics of Taste Lab explored this apparent inconsistency in counting and found that scorers were individually prioritizing the importance of these characteristics differently and had no guidance for when a papilla had some, but not all, of the reported qualities of FP. The result of this subjectivity is highly variable FP counts of the same tongue image. The Denver Papillae Protocol has been developed to remedy this consequence through use of a dichotomous key that further defines and prioritizes the importance of the characteristics put forth by Miller &#38; Reedy. The proposed method could help create a standard way to quantify FP for researchers in the field of taste and nutritional studies.

Here is a representative photograph scored by two individual scorers when using the commonly-used Miller &#38; Reedy methodology for FP identification based on the field standards of round, large, pink or stained lighter, and elevated, and then the same photograph scored by two scorers using DPP. The counts shown are representative of the high variance observed using the original method compared to the lower variance using DPP.
<img alt="Figure 4" src="/files/ftp_upload/52860/52860fig4.jpg" />
Figure 4: Miller &#38; Reedy Quantification vs DPP Quantification. The image on the left is representative of two scorers&#8217; counts using the Miller &#38; Reedy Method. The image on the right represents two scorers&#8217; counts using DPP. Red is scorer 1, Yellow is scorer 2, Green is scorers 1 &#38; 2.
Statistical reliability of DPP was previously studied using a mixed linear model to assess variability10. Briefly, to generate data to assess the role DPP plays in consistency of scores, fifteen images were scored twice. In the first batch of scores, DPP na&#239;ve scorers used the Miller&#38; Reedy method2. The scorers were then trained to use DPP and the images were individually rescored without the final consensus step described in section 2.7 detailing Quality Control (Figure 5). The FP score data set was then used in the mixed model to assess the combined differences due to protocol (Figure 6), within-scorer variability and the interaction between the two. This model showed a significant difference in scoring when using the Miller &#38; Reedy method compared to DPP (p-value &#60;1 x 10-6) with DPP leading to a higher count by 6.99 (SE = 0.99); 5.2% of the variability in the score was due to training, 25.9% due to scorer, and the remaining due to variability specific to the image10. Next, to generate data to determine within-scorer variability, a series of thirty images in random order were scored by 11 individuals. Unbeknownst to scorers, this series of images included three images that were repeated. We observed a drastic difference in score variability comparing distinct images versusrepeated images.
<img alt="Figure 5" src="/files/ftp_upload/52860/52860fig5.jpg" /> 
Figure 5: DPP-Na&#239;ve vs Post DPP Training. Panels provide boxplots of reviewer scores for 15 images pre and post Denver Papillae Protocol training. For each boxplot, a filled in circle indicates the mean whereas an open circle indicate an outlier.
<img alt="Figure 6" src="/files/ftp_upload/52860/52860fig6.jpg" /> 
Figure 6: Change in Scorer Variance. The gray represents the variance for individual scorers prior to DPP training. Black is the same scorer post DPP training.

Watch this Scientific Journal Video about Denver Papillae Protocol for Objective Analysis of Fungiform Papillae at JoVE.com

Denver Papillae Protocol for Objective Analysis of Fungiform Papillae

Here we present a protocol to measure fungiform papilla density from digital photographs. This method builds prioritization and objective characteristic metrics into the original descriptive work on fungiform papillae by Miller &#38; Reedy (1990).

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure ...

denver-papillae-protocol-for-objective-analysis-of-fungiform-papillae

Research

JoVE Journal

Neuroscience

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 ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual&#39;s oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual&#39;s ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.

Oral chemoreception of fatty acids and the association with diet and fatty food preferences may enable the identification of mechanisms involved with the development of obesity and why dietary changes may be difficult for many individuals.

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

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 ...

Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the analysis of density and size gradients of retinal cell types, the direction tuning of direction-selective ganglion cells, and the examination of topographic degeneration patterns in some retinal diseases. However, there are many different ocular dissection methods reported in the literature that are used to identify and label retinal orientation in the mouse retina. While the method of orientation used in such studies is often overlooked, not reporting how retinal orientation is determined can cause discrepancies in the literature and confusion when attempting to compare data between studies. Superficial ocular landmarks such as corneal burns are commonly used but have recently been shown to be less reliable than deeper landmarks such as the rectus muscles, the choroid fissure, or the s-opsin gradient. Here, we provide a comprehensive guide for the use of deep ocular landmarks to accurately dissect and document the spatial orientation of an isolated mouse retina. We have also compared the effectiveness of two s-opsin antibodies and included a protocol for s-opsin immunohistochemistry. Because orientation of the retina according to the s-opsin gradient requires retinal reconstruction with Retistruct software and rotation with custom code, we have presented the important steps required to use both of these programs. Overall, the goal of this protocol is to deliver a reliable and repeatable set of methods for accurate retinal orientation that is adaptable to most experimental protocols. An overarching goal of this work is to standardize retinal orientation methods for future studies.

This protocol provides a comprehensive dissection and analysis guide for the use of deep ocular landmarks, s-opsin immunohistochemistry, Retistruct, and custom code to accurately and reliably orient the isolated mouse retina in anatomical space.

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...