We thank Joachim Erber for introducing us to the tactile learning paradigm in honey bees. This work was supported by the Cluster of Excellence 277 CITEC, funded in the framework of the German Excellence Initiative.

1. Preparing the Bees
<ol>
	<li>Nectar or Pollen foragers are caught in the field either from a sucrose feeder or directly from the hive entrance while returning from a foraging trip. Each single bee is captured into a glass vial that is closed with a foam plug and taken immediately into the laboratory for further handling.</li>
	<li>In the laboratory, the captured bees are briefly cooled in the refrigerator at 4 &deg;C until they show first signs of immobility.</li>
	<li>Each single immobilized bee is mounted in a small metal tube with adhesive tape between head and thorax and over the abdomen. Care should be taken that the proboscis and antennae are freely movable.</li>
	<li>Paint the compound eyes and ocelli of the fixed bee with white paint (e.g. solvent-free Tipp-Ex) to occlude vision.</li>
	<li>Add a small drop of melted wax behind the head of the bee to fix it to the tape between head and thorax to prevent head movements during recordings.</li>
	<li>Mark each single bee with a number on the tape for better identification and place the tube with the fixed bee into a humid atmosphere to prevent dehydration.</li>
	<li>Feed each single bee for 5 sec with droplets of a 30% sucrose solution presented with a syringe and let all bees recover for 30 min before starting with the tactile conditioning protocol.</li>
</ol>
2. Tactile Conditioning
<ol>
	<li>Before conditioning, each single bee has to be tested for the proboscis extension response (PER) to a 30% sucrose stimulus applied to the antennae. Thereby the tip of the proboscis has to cross a virtual line between the opened mandibles. Discard all bees that don't respond with a PER to the sucrose stimulus.</li>
	<li>For tactile conditioning use a brass cube (e.g. 3 x 5 mm) with a smooth or an engraved pattern, e.g., horizontal or vertical grooves forming a grating with 150 &mu;m wave length, as the conditioned stimulus (CS). For the unconditioned stimulus (US) use a 30% sucrose solution (household sugar, diluted in water).</li>
	<li>The brass cube (CS) is placed into a holder on a micromanipulator (e.g., M&auml;rzh&auml;user MM33) to ensure exact positioning during the conditioning procedure. The US is presented to the bee with a syringe filled with a 30% sucrose solution.</li>
	<li>The conditioning procedure consists of five pairings of the tactile stimulus (CS) and sucrose solution (US) with an inter-trial interval (ITI) of 5 min. Place a single bee in front of the micromanipulator with the mounted tactile stimulus (CS). Position the CS slowly, such that the surface of the tactile stimulus is parallel to the head of the bee (Figure 1 A and B). The distance between the animal and the tactile object should be in the range of the antennal working radius of the tested bee, i.e. the bee should be able to scan the tactile stimulus in a comfortable position with both antennae. In the example shown in Figure 1, the distance was 3 mm. Let the bee scan the tactile stimulus (CS) for 5 sec. After the first 3 sec, present a droplet of the 30% sucrose (US) solution with a syringe under the proboscis. Use the tip of the syringe to gently raise the proboscis. Sucrose stimulation under the proboscis will elicit the unconditioned PER15. Allow the bee to lick the sucrose reward. Use a stopwatch with an alarm signal to maintain the exact time intervals during conditioning.</li>
	<li>The conditioned PER is used as a measure for the learning success of a bee. After the first rewarded pairings, bees start to respond to CS presentation by extension of their proboscis, indicating that they expect the forthcoming reward. A fully extended proboscis observed anytime during the 3 sec time window of tactile stimulus presentation and before sucrose presentation is scored as a positive response. No response is counted negative. The occurrence of the PER has to be noted by the experimenter. The percentage of bees showing the PER during CS presentation is plotted for each trial.</li>
</ol>
3. Kinematic Recordings
<ol>
	<li>Antennal movements of a single harnessed bee are recorded with a digital video camera with a suitable macro lens (e.g. Basler A602f-2 equipped with TechSpec VZM 200, operated at 50 fps via a fire wire connection). The camera is positioned above the animal in a top-down view (Figure 1 A). In Figure 1, the spatial resolution of the recordings is 0.02 mm/pixel.</li>
	<li>Calibrate the camera by recording single pictures of a 10 x 10 mm checkerboard with an edge length of 1 mm from different orientations under the camera objective. The calibration can be done with the camera calibration tool box Matlab16.</li>
	<li>Place a single fixed bee below the camera lens. Present the tactile stimulus, fixed to a micromanipulator, to the animal. Proceed in the same way as described for tactile conditioning, and record the antennal movement while the bee is scanning the object. It is important that the full antennal working range and the tactile stimulus are visible. The choices of stimulus patterns tested and trial numbers depend on the experimental design.</li>
</ol>
4. Data Analysis
<ol>
	<li>The computation of the image background is done in Matlab. First, the median greyscale value over time has to be calculated for each pixel. Static objects, like the fixed head of the bee and the tactile stimulus, will constitute the image background. Moving objects, like the two antennae, will not be part of the background.</li>
	<li>Each frame of the recorded video has to be loaded sequentially, and the difference between the current frame and the image background has to be computed. The result of this subtraction emphasizes those parts of the image that are moving from frame to frame. Ideally, the antennae of the bee are the only areas of non-zero values (Figure 1C).</li>
	<li>For further processing, the two largest areas with non-zero pixel values are assumed to be the antennae. For both antennae a binary mask is to be generated containing for each pixel a value 1, if the pixel belongs to the antenna and value 0 otherwise. This mask serves as a basis for localizing the antennal tips later. To gain a preliminary mask for the entire image the grey-value of each pixel of the difference image is compared to a pre-defined threshold. Since we take care of noise later this threshold is chosen to be quite low. The preliminary mask then still carries two kinds of errors: Firstly, due to image noise small regions are still part of the mask. Secondly, areas that belong to the antenna might not necessarily be fully connected. The latter mostly occurs if the background has approximately the same luminescence as the antenna. To eliminate these artifacts, standard image processing morphological operation are applied, i.e. a combination of image erosion and dilatation, relying upon the standard Matlab functions imerode and imdilate respectively (see17 pp. 158-205 for further explanation). After the denoising process the antennae hypotheses are still contained within the same mask. Hence, as a next step the binary mask is clustered for disjoint areas using the standard Matlab function bwlabel (see17 pp. 40-48 for further details on the image segmentation algorithm).</li>
	<li>The number of pixels per cluster is counted and the two largest clusters are selected. The center of gravity is calculated to distinguish between the left and right antenna (Figure 1D).</li>
	<li>The antennal tip for each antenna can be defined as the pixel in a cluster with the highest value in the proximal-to-distal direction (see Figure 1D).</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The wisdom of the hive. , (1995).</li><li>Staudacher, E., Gebhardt, M. J., D&#252;rr, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+movements+and+mechanoreception:+neurobiology+of+active+tactile+sensors.">Antennal movements and mechanoreception: neurobiology of active tactile sensors.</a> Adv. Insect Physiol. 32, 49-205 (2005).</li><li>Martin, H., Lindauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinnesphysiologische+Leistungen+beim+Wabenbau+der+Honigbiene.">Sinnesphysiologische Leistungen beim Wabenbau der Honigbiene.</a> Z. Vergl. Physiol. 53, 372-404 (1966).</li><li>Kevan, P. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+learning+and+the+individual+evaluation+of+the+reward+in+honey+bees+(Apis+mellifera+L.).">Tactile learning and the individual evaluation of the reward in honey bees (Apis mellifera L.).</a> J. Comp. Physiol. A. 185, 1-10 (1999).</li><li>Erber, J., Scheiner, R., Adelman, G., Smith, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honeybee+learning.">Honeybee learning.</a> Encyclopedia of Neuroscience. , (2004).</li><li>Bitterman, M. 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W., Woodring, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+stress,+age,+season+and+source+colony+on+levels+of+octopamine,+dopamine+and+serotonine+in+the+honey+bee+(Apis+mellifera+L.)+brain.">Effects of stress, age, season and source colony on levels of octopamine, dopamine and serotonine in the honey bee (Apis mellifera L.) brain.</a> J. Insect. Physiol. 38, 29-35 (1992).</li><li>Scheiner, R., Kuritz-Kaiser, A., Menzel, R., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+responsiveness+and+the+effects+of+equal+subjective+rewards+on+tactile+learning+and+memory+of+honeybees.">Sensory responsiveness and the effects of equal subjective rewards on tactile learning and memory of honeybees.</a> Learn. Mem. 12, 626-635 (2005).</li><li>Mujagic, S., Sarkander, J., Erber, B., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sucrose+acceptance+and+different+forms+of+associative+learning+of+the+honey+bee+(Apis+mellifera+L.)+in+the+field+and+laboratory.">Sucrose acceptance and different forms of associative learning of the honey bee (Apis mellifera L.) in the field and laboratory.</a> Front. Behav. Neurosci. 4, 46 (2010).</li><li>Scheiner, R., Barnert, M., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+water+and+sucrose+responsiveness+during+the+foraging+season+affects+proboscis+extension+learning+in+honey+bees.">Variation in water and sucrose responsiveness during the foraging season affects proboscis extension learning in honey bees.</a> Apidologie. 34, 67-72 (2003).</li><li>Mujagic, S., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sucrose+acceptance,+discrimination+and+proboscis+responses+of+honey+bees+(Apis+mellifera+L.)+in+the+field+and+the+laboratory.">Sucrose acceptance, discrimination and proboscis responses of honey bees (Apis mellifera L.) in the field and the laboratory.</a> J. Comp. Physiol. 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No conflicts of interest declared.

Preparation of bees: Collecting and fixing the bees should be done quickly, in order to keep the stress level of the animal low. Stress has an effect on the PER-responsiveness and, therefore, could have an indirect effect on the learning performance in bees19, 20. The stress level can be decreased by placing the glass vials with the bees directly on ice immediately after collection to immobilize them quickly. It has to be taken into account that bees need more time to recover the longer the anesthesia period is before.
Vision has a negative effect on tactile learning in bees18. Therefore it is important to occlude vision by painting the eyes of the animal. In order to increase the visual contrast of the video recordings, it is recommended to use white paint (e.g., Tipp-Ex). Care should be taken not to get the antennae in contact with the paint.
Learning performance: Former studies in the laboratory and field showed that sucrose sensitivity in honey bee foragers affect their associative learning ability21, 22. High sucrose sensitivity is positively correlated with better tactile acquisition and discrimination performances. Pollen foragers of a honey bee colony are on average more responsive to sucrose than nectar foragers in most weeks of a foraging season23. They can be easily identified by the pollen loads on their hind legs and caught at the hive entrance. Nectar foragers with high sucrose sensitivity can be caught at the end of a foraging season, between September and October. They can be selected on artificial feeders filled with low-concentrated sugar water placed approximately 20 m away from the hive23.
The learning performance and memory formation depends strongly on the satiation status of a bee during conditioning25. A longer starvation period before conditioning, e.g. overnight, can improve the associative learning behavior and increase the motivation level of an animal.
Tactile conditioning: The PER is used as the unconditioned response in an appetitive learning protocol14. Therefore, it is crucial that all tested bees are showing a fully extended PER to the unconditioned stimulus (sugar stimulus) before conditioning. Bees that do not respond to the sugar stimulus should be discarded as well as bees that respond with a PER during antennal contact with the tactile stimulus before conditioning. Otherwise, the extremely low or extremely high responsiveness of these animals will confound the learning curve.
The tactile stimulus used in this protocol (brass cube with horizontal gratings) was randomly chosen. Honey bees can be also conditioned on different tactile stimuli with various shapes and textures (see10).
The tactile stimulus must be cleaned after each conditioning trial and after contact with the tongue of an animal by immersing it in 70% ethanol and drying it with a cellulose cloth.
To compare acquisition in different groups of bees (e.g. Pollen- versus Nectar foragers), an acquisition score can be calculated21. This score is the total number of conditioned responses to the CS and it has a range between 0 (no conditioned PER) and 5 (conditioned PER after each conditioning trial).
Video analysis: Different video analyses methods were used in the past to quantify changes in the antennal movement behavior in honey bees26, 27. Our method has the advantage of analyzing the antennal movements without requiring reflective markers on the antennae and benefits from the variety of image processing tools in Matlab. As an example, one can also analyze the timing of antennal contacts towards an object, determine the speed of antennal movement, calculate the angular positions of the antennae in reference to the body axes or discriminate between left and right antennal movement.
A PER that occurs during recording of antennal response to the object in conditioned bees could interfere later with the automatic detection of the antennae. In this case it is necessary to edit the movie, e.g. in VirtualDub, and to mask the object that interferes with the detection process, e.g. by covering the section where the proboscis appears with a grey layer of the same shape.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Pollen foragers, Apis mellifera L.</td>
 <td></td>
			<td></td>
			<td>Private hives</td>
		</tr>
		<tr>
			<td>Glass vials (22 x 45 mm)</td>
			<td>Carl Roth GmbH &amp; Co</td>
			<td>X655.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Brass tubes, 10 x 30 mm</td>
			<td></td>
			<td></td>
			<td>Self-produced</td>
		</tr>
		<tr>
			<td>Brass cube, 3 x 5 mm</td>
			<td></td>
			<td></td>
			<td>Self-produced</td>
		</tr>
		<tr>
			<td>Tipp-Ex (solvent-free)</td>
			<td>Tipp-Ex GmbH &amp; Co KG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose solution (30%)</td>
			<td></td>
			<td></td>
			<td>Household suger diluted in water</td>
		</tr>
		<tr>
			<td>Ethanol (70%)</td>
			<td>Carl Roth GmbH &amp; Co</td>
			<td>9065.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (1 ml) and needle (0.6 x 25)</td>
			<td>Carl Roth GmbH &amp; Co</td>
			<td>59.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Stop Watch</td>
			<td>Carl Roth GmbH &amp; Co</td>
			<td>L423.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>M&auml;rzh&auml;user MM33</td>
			<td>00-42-101-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital video camera</td>
			<td>Basler A602f-2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Macro lens for camera</td>
			<td>TechSpec VZM 200</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab R2009b</td>
			<td>The MathWorks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

tactile conditioning and movement analysis of antennal sampling strategies in honey bees (apis mellifera l.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood3. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks4, including hive building5 and pattern recognition6. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates7. Bees use different floral signals like colors or odors7, 8, but also tactile cues from the petal surface9 to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae10, 11, 12, 13. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees14. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

In the following experiment we studied how tactile learning affects antennal sampling behavior in honey bees. For this, we monitored the movement of the antennal tip in na&iuml;ve and conditioned bees before and during presentation of a tactile stimulus.
First, the spontaneous antennal movement of a group of pollen foragers (N = 42) was recorded for 1 min. One half of the bees (N = 21) was then conditioned by pairing five times a tactile stimulus with a 30% sucrose reward. This was the conditioned group. The tactile stimulus was the 30 x 50 mm surface of a brass cube with an engraved horizontal grating ( &lambda;= 150 &mu;m). The other half of the bees (N = 21) was not conditioned and used as a na&iuml;ve group. Figure 2 shows the mean learning curve of the conditioned group of bees. The percentage of conditioned PER after the first reward is significantly different from the spontaneous behavior (Figure 2, Fisher exact probability test, p &lt; 0.001). The learning curve shows saturation after the 3rd reward.
Afterwards the movement of the antennal tip in both the conditioned and the na&iuml;ve group was recorded again for 1 min in presence of the tactile stimulus. Figure 3 A1 - B2 shows the distribution of antennal tip locations in both groups, before and during presentation of the tactile object. Both, the na&iuml;ve and the conditioned group changed their antennal movement pattern in presence of a tactile stimulus compared to the spontaneous behavior. The likelihood of the antennal tip to search and/or sample the area of the stimulus is significantly increased if the stimulus was present, showing that tactile sampling involves a marked change in the antennal movement pattern compared to spontaneous antennal movement. This is true for both groups of bees (Figure 3 C1 and C2, Wilcoxon signed-rank test, P &lt; 0.001 in both cases). Bees from the conditioned group spent significantly more time with their antennal tip in the area of the stimulus than the na&iuml;ve bees (Figure 3 C1 and C2, Mann-Whitney U test, P &lt; 0.05; there was no significant difference between the two reference periods A in Figure 3C). Thus, tactile conditioning leads to an increase of active antennal movement near the object and tactile sampling of the object surface as previously shown in Erber (2012)18. Compared to the na&iuml;ve group, the spatial distribution during stimulus presentation was much smoother after conditioning (Compare Figure 3 B1 and B2).
In summary, the results from this experiment show that honey bees can be trained very effectively to respond to a tactile stimulus, and that tactile learning is accompanied by increased active searching and sampling behavior.
<img alt="Figure 1" fo:content-width="5.5in" fo:src="/files/ftp_upload/50179/50179fig1highres.jpg" src="/files/ftp_upload/50179/50179fig1.jpg" /> 
 Figure 1. Video recording and antennal tracking procedure. A: Top-down view of a fixed bee and the tactile stimulus. The tactile stimulus is positioned in front of the head of the fixed bee with occluded eyes. It is located within the antennal working- range. d and p indicate distal and proximal locations relative to the animal. B: Side view of the same bee and the tactile stimulus. The surface of the tactile stimulus and the head of the bee (blue dotted lines) are parallel to each other. C: Distinguishing moving pixels from static pixels in a video sequence. Subtracting each image from a background model allows identification of the moving parts, i.e., the antennae. The fixed head of the bee and the tactile stimulus (indicated here as red dotted lines) are static and not visible after the subtraction. D: Defining left and right and the antennal tip. The two largest pixel clusters after subtraction are identified as antennae. Calculation of the center of gravity allows distinguishing between the left (red) and the right (green) antenna. The antennal tip for each antenna (black cross) can be defined as the pixel in a cluster with the largest value in the distal direction.
<img alt="Figure 2" src="/files/ftp_upload/50179/50179fig2.jpg" /> 
 Figure 2. Learning curve of tactile PER conditioning. Pollen foragers were trained using five pairings of the CS (tactile stimulus) and US (sucrose solution) with an inter-trial interval of 5 min. The percentage of positive responses of 21 animals is plotted for the spontaneous behavior (spont) and during presentation of the CS after each conditioning trial. The percentage of PER observed after the first and all following rewards is significantly different from the spontaneous behavior (Fisher exact probability test, p &lt; 0.001).
<img alt="Figure 3" fo:content-width="4.8in" fo:src="/files/ftp_upload/50179/50179fig3highres.jpg" src="/files/ftp_upload/50179/50179fig3.jpg" /> 
 Figure 3. Normalized average distributions of the antennal tip location during a period of 1 min. The antennal tip distribution of each bee was normalized to the total number of data points per animal. The panels show results from na&iuml;ve (left, N = 21) and conditioned bees (right, N = 21) before (A) and during stimulus presentation (B). A1 and A2: Spontaneous antennal movement before stimulus presentation. The abscissa shows the lateral position of the antennal tip relative to the center of the head (compare with Figure 1C and D). The ordinate shows the distal position of the antennal tip. The color scale indicates the percentage of time that the antennal tip spent at any given location. The recording period of 1 min was equivalent to 3,000 video frames. B1 and B2: Same as in A1,2, but during stimulus presentation. C1 and C2: Average number of frames in which the antennal tip was located inside the object area, i.e., &plusmn; 3 bins distal or proximal relative to the stimulus surface. C1: na&iuml;ve group; C2: conditioned group. The box-whisker plots show the median, mean, quartiles, and 5-95% percentile ranges of number of frames per minute. In both groups of bees, there is a significant difference between 'no stimulus' (A) and 'with stimulus' (B, C; Wilcoxon signed-rank test, C1: P = 5.39*10-4; C2: P = 5.96*10-5). The average number of frames during tactile stimulus presentation is significantly larger in conditioned bees than in na&iuml;ve bees (Mann-Whitney U test, C1 - C2: P = 0.007). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50179/50179fig3large.jpg" target="_blank"> Click here to view larger figure</a>.

Watch this Scientific Journal Video about Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L. at JoVE.com

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

tactile-conditioning-movement-analysis-antennal-sampling-strategies

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.5K Views.  Bielefeld University. In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

Video: Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

Eye Movement Monitoring of Memory

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank yesterday."), however such reports can often be unreliable or sensitive to response bias 1, and may be unobtainable in some participant populations. Furthermore, explicit reports only reveal when information has reached consciousness and cannot comment on when memories were accessed during processing, regardless of whether the information is subsequently accessed in a conscious manner. Eye movement monitoring (eye tracking) provides a tool by which memory can be probed without asking participants to comment on the contents of their memories, and access of such memories can be revealed on-line 2,3. Video-based eye trackers (either head-mounted or remote) use a system of cameras and infrared markers to examine the pupil and corneal reflection in each eye as the participant views a display monitor. For head-mounted eye trackers, infrared markers are also used to determine head position to allow for head movement and more precise localization of eye position. Here, we demonstrate the use of a head-mounted eye tracking system to investigate memory performance in neurologically-intact and neurologically-impaired adults. Eye movement monitoring procedures begin with the placement of the eye tracker on the participant, and setup of the head and eye cameras. Calibration and validation procedures are conducted to ensure accuracy of eye position recording. Real-time recordings of X,Y-coordinate positions on the display monitor are then converted and used to describe periods of time in which the eye is static (i.e. fixations) versus in motion (i.e., saccades). Fixations and saccades are time-locked with respect to the onset/offset of a visual display or another external event (e.g. button press). Experimental manipulations are constructed to examine how and when patterns of fixations and saccades are altered through different types of prior experience. The influence of memory is revealed in the extent to which scanning patterns to new images differ from scanning patterns to images that have been previously studied 2, 4-5. Memory can also be interrogated for its specificity; for instance, eye movement patterns that differ between an identical and an altered version of a previously studied image reveal the storage of the altered detail in memory 2-3, 6-8. These indices of memory can be compared across participant populations, thereby providing a powerful tool by which to examine the organization of memory in healthy individuals, and the specific changes that occur to memory with neurological insult or decline 2-3, 8-10.

Eye movement monitoring (or eye tracking) reveals where in space the eyes linger, when and for how long. Here, we demonstrate how eye tracking can be used to investigate the integrity of memory in multiple participant populations, without requiring verbal, or otherwise explicit, reports.

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank ...

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16.
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and focused on psychological aspects. Until now, objective observation of neurophysiological parameters has not been carried out - undoubtedly because the technical and methodological means have not been available -, investigations into the neurophysiological effects of weightlessness are in their infancy (Schneider et al. 2008).
While imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) would be hardly applicable in space, the non-invasive near-infrared spectroscopy (NIRS) technique represents a method of mapping hemodynamic processes in the brain in real time that is both relatively inexpensive and that can be employed even under extreme conditions. The combination with electroencephalography (EEG) opens up the possibility of following the electrocortical processes under changing gravity conditions with a finer temporal resolution as well as with deeper localization, for instance with electrotomography (LORETA).
Previous studies showed an increase of beta frequency activity under normal gravity conditions and a decrease under weightlessness conditions during a parabolic flight (Schneider et al. 2008a+b). Tilt studies revealed different changes in brain function, which let suggest, that changes in parabolic flight might reflect emotional processes rather than hemodynamic changes. However, it is still unclear whether these are effects of changed gravity or hemodynamic changes within the brain. Combining EEG/LORETA and NIRS should for the first time make it possible to map the effect of weightlessness and reduced gravity on both hemodynamic and electrophysiological processes in the brain. Initially, this is to be done as part of a feasibility study during a parabolic flight. Afterwards, it is also planned to use both techniques during medium- and long-term space flight.
It can be assumed that the long-term redistribution of the blood volume and the associated increase in the supply of oxygen to the brain will lead to changes in the central nervous system that are also responsible for anaemic processes, and which can in turn reduce performance (De Santo et al. 2005), which means that they could be crucial for the success and safety of a mission (Genik et al. 2005, Ellis 2000).
Depending on these results, it will be necessary to develop and employ extensive countermeasures. Initial results for the MARS500 study suggest that, in addition to their significance in the context of the cardiovascular and locomotor systems, sport and physical activity can play a part in improving neurocognitive parameters. Before this can be fully established, however, it seems necessary to learn more about the influence of changing gravity conditions on neurophysiological processes and associated neurocognitive impairment.

The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. 
A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and ...

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

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

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

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 ...

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor learning. There exists a number of noninvasive electrophysiological methods investigating the excitability and plasticity of this pathway. However, most methods are based on quantification of compound potentials and neglect that the corticospinal pathway consists of many different connections that are more or less direct. Here, we present a method that allows testing excitability of different fractions of the corticospinal transmission. This so called H-reflex conditioning technique allows one to assess excitability of the fastest (monosynaptic) and also polysynaptic corticospinal pathways. Furthermore, by using two different stimulation sites, the motor cortex and the cervicomedullary junction, it allows not only differentiation between cortical and spinal effects but also assessment of transmission at the corticomotoneural synapse. In this manuscript, we describe how this method can be used to assess corticomotoneural transmission after low-frequency repetitive transcranial magnetic stimulation, a method that was previously shown to reduce excitability of cortical cells. Here we demonstrate that not only cortical cells are affected by this repetitive stimulation but also transmission at the corticomotoneuronal synapse at the spinal level. This finding is important for the understanding of basic mechanisms and sites of neuroplasticity. Besides investigation of basic mechanisms, the H-reflex conditioning technique may be applied to test changes in corticospinal transmission following behavioral (e.g., training) or therapeutic interventions, pathology or aging and therefore allows a better understanding of neural processes that underlie movement control and motor learning.

The aim of the present study was to assess changes in transmission at the corticomotoneuronal synapses in humans after repetitive transcranial magnetic stimulation. For this purpose, an electrophysiological method is introduced that allows assessment of pathway specific corticospinal transmission, i.e. differentiation of fast, direct corticospinal pathways from polysynaptic connections.

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor ...

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

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Eye Movement Monitoring of Memory

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders