The purpose of this document is to standardize the training protocols for Proboscis Extension Response conditioning of honey bees. Anyone performing PER studies should be aware of how variations in the training procedure can affect the results. Indeed it is impossible to completely standardize the protocol across all experiments and researchers, because many times experimental manipulations require that the protocol be changed. Nevertheless, there are aspects of the procedure that need to be used in the proper way in order to minimize the possibility of artifact. At least then any variation in the procedure would change as few variables as is necessary across experimenters. The document that follows is meant as a beginner's guide to the PER protocols. It was last updated on 3-May-1998.

CONSTANCY

ODOR SETUP

COLLECTION AND SETUP OF DRONES, WORKERS AND QUEENS

DELIVERY OF THE CS

DELIVERY OF THE US
 
RECORDING RESPONSES

EXAMPLES OF VIDEOTAPED RESPONSES TO CONDITIONED ODORS

CONDITIONING AND TEST TRIALS

MISCELLANEOUS STUFF

EQUIPMENT YOU'LL NEED
 

CONSTANCY

• Whatever procedure you settle on, run it by others in the laboratory for comments
• DO NOT vary this procedure within your experiment
• if you do, the results may not be statistically comparable!!!

ODOR SETUP

• Make sure that odor does not get on your hands during the procedure
• wear gloves if necessary
• thoroughly wash your hands afterwards
• We us 1cc glass tuberculin syringes for odor cartridges and automate odor delivery via the PIO12 program (see below)
• we no longer use manual or automated delivery from 20 cc plastic syringes
• Make sure the 1cc glass syringe is clearly labeled in regard to the odor for which it has been used
• an odor is defined as the volatile pattern given off by an odorant (pure) or a mixture
• label in writing and by color:  1-hexanol (green); geraniol (yellow); 2-octanone (blue); hexanal (white); all mixtures (red); everything else (orange)
• Rinse the inside of the 1 cc glass tuberculin syringe with alcohol and wash off the black rubber restrictor that goes into the end of each syringe
• Set up the syringe on the ledge of the exhaust hood
• never open jars of odor anywhere but in front of this hood in room 6
• Place a small strip of filter paper into the tube such that the end sticks out about 1-2 cm
• Place 3 ml of pure odorant or 5 ml of a dilution in solvent onto the filter paper and then discard the tip of the Pipetteman before setting up the next odor
• do not let the odor touch the end of the tube!
• the Pipetteman tip can be reused for the SAME odorant, but never stick it into two different odors
• Turn the syringe upward so that the filter paper slides rapidly into the tube
• Thoroughly wash the tube with alcohol after setup to prevent any odor from tip from reaching the bee
• ball up the end of a Kimwipe soaked in alcohol and make sure to thorougly wash the black plug in addition to the outside of the glass part of the syringe
• GENERAL RULES:
• IF IN DOUBT about prior usage of a syringe, get a new syringe and discard the old one
• NEVER EVER EVER EVER EVER mix tubes or plugs; that is, the same plug should always go into the same tube
• once a syringe has been used for a particular odorant or mixture it can only be used for that odor REGARDLESS of how much it has been cleaned
• Setup up all of the different odors you’ll need for acquisition, and setup a NEW set of syringes for TEST trials

COLLECTION AND SETUP OF DRONES, WORKERS AND QUEENS

• WORKERS should be collected from the entrance or from frames by placing a scintillation vial over them until they fly into it
• make sure the lid has a hole in it!
• only one bee/vial
• place the vials into an ICE/WATER SLURRY until the bee inside stops moving
• remove immediately to avoid overexposure to cold and place the bee into a harness
• DRONES should be collected in vials  late in the day as they return from mating flights
• put an excluder in front of the entrance and let returning drones collect on it
• place them into an excluder cage
• put the cage into a cell builder overnight
• make sure there is space between the cage and the frames so that nurse bees can enter the cage to feed to drones
• early the next day place the cage into a small chamber and remove the plug
• place the drones directly into harnesses
• QUEENS should be removed from cages and placed directly into restraining harnesses
• Make sure the tape on the harness is tight
• too much space and the bee will escape or frequently not be able to extend its proboscis properly
• There are two different times to do the setup
• use one for each experiment!!
• don’t switch between them within an experiment
• you may need to use one or the other depending on whether you’re using flightroom or outside bees, queens/drone/workers, etc.
• if you’re comparing different groups of bees THEY NEED TO BE TREATED IN THE SAME WAY!!!
• METHOD 1:  SETUP ON THE DAY OF TESTING:
• setup bees in harnesses the first thing in the morning
• between 30 min and 1 hr, no more, after setup feed all bees 0.4 ml of the sucrose to be used for the US on that day
• allow at least 1 hr (preferably 2 hrs) beyond this point for recovery before training
• METHOD 2:  SETUP ON THE DAY BEFORE TESTING
• make sure the setup procedure is COMPLETE at least by 24 hr before testing is to begin
• between 30 min and 1 hr, no more, after setup feed all bees 0.4 ml of the sucrose to be used for the US during training
• feed on several different passes through a set of bees until the bees are satiated
• that is, until the proboscis is no longer extended by antennal contact with sucrose
• place bees on a countertop overnight
• do not feed them the next morning
• 24 hr after feeding begin the training protocol
• note that we generally find poorer conditioning performance with this protocol, which probably stems from lower overall motivational states of the bees
• CAUTION:  preexposure to the US can influence subsequent conditioning!
• therefore feed in an area well removed from the training locale

• The PIO12 program is used to time the delivery of odors and signal delivery of the US
•    see EQUIPMENT YOU'LL NEED if you would like to obtain a free copy of this program
• The program will prompt you for a <filename>.stm file when it begins
• this file lists the different delivery protocols (up to 9) for each experiment
• make sure you are aware of the contents of the specific file you are using!
• At the beginning of each day, and maybe even at intervals throughout each day, check the electrical and air connections
• you can check air delivery by placing a moist fingertip in front of the top outlet of the white valve on the relay board
• if you don’t feel a strong air stream, then something’s wrong
• check the exhaust system in the same way to make sure the tubing has not come apart
• also, if the electrical connection is broken for some reason, the computer will work but the board and/or the valve will not
• make sure the LED lights work on a trial run of the system before you begin each day
• also, if it’s not working, check to make sure that the switches underneath the table in front of the hood are on and that the aquarium pumps are on

• Adjust the odor syringe in the clay holder so that it points directly at the bee’s antennae
• use a small plexiglass stand on top of which some modeling clay is placed
• the glass odor syringe can be placed in the clay and adjusted to point at the bee's head
• Securely place the fitting connecting the syringe to the valve over the ground glass end of the syringe
• Position the end of the syringe 1-2 cm from the bee
• no farther than 2 cm and not closer than 1!!
• pick a distance within this range and be constant about it
• If the bee begins to respond immediately to the syringe, try to smell odor on the tip
• if you can smell some when the other end is closed by the fitting, wash it off with a Kimwipe soaked in 95% ethanol
• if you continue to have trouble with the syringe throw it out and get a new one
• The air flow rate is on the order of 400 ml/min coming in from the aquarium pumps
• it is good to check this flow rate periodically
• regularly (daily) make sure the valve is switching properly by placing the tube coming out from the valve into a vial of water or hooking it up to a flowmeter
• 400 ml/min means that about 7 ml/sec are pushed through the odor cartridge; so over 4 sec 28 ml will be pushed through a 1 ml cartridge

• Load the Gilmont syringe with the sucrose solution so that there are no bubbles in the glass portion and with the micrometer wound back as far as possible
• Wind the micrometer forward a turn or two to make sure that the fluid in the needle is actually sucrose and not water from a wash
• Turn the micrometer to deliver a multiple of 0.2 ml, which is the smallest increment on the syringe
• we have fed a number of ways in the past, but it should from now on be standardized
• different protocols will require different amounts of feeding
• generally we should be working in the 0.2, 0.4, 0.6 and 0.8 ml range
• DO NOT use “ad libitum” feeding, which is a large droplet that the bee feeds from for several seconds and cannot entirely consume
• Hold the syringe approximately 2 cm from the bee as odor is presented
• DO NOT get it too close in anticipation of the feeding signal!!!!!
• use the edge of the training arena to balance your hand so that it does not shake
• When the feeding signal is delivered by the computer LIGHTLY touch the antennae until the proboscis extends, then feed the bee
• this should require only a very light touch to the antennae
• watch out for sucrose build up on the antenna
• DO NOT GET THE SUCROSE TOO CLOSE BEFORE THE FEEDING SIGNAL OCCURS, and make sure you’re there with it as soon after the beep as is possible
• If you are using up to 0.8 µl the bee should easily consume the entire droplet within the time period alloted for the US
• Watch for buildup of sucrose on the antennae and modify your procedure if you see it!

• A response (‘+’) is defined as anytime that the bee extends its proboscis beyond the line made by connecting the tips of the opened mandibles
• stay away from trying to score partial responses - if you need to make finer distinctions use the video technique outlined below
• only score a ‘+’ when the behavior begins with or after the onset of odor
• if the proboscis i never extended, or is extended after the offset of odorant, then it is not a response (i.e., a ‘-’)
•  Scoring responses as either a + or a - is satisfactory for many kninds of analyses
• treatment groups pf 20-40 bees can be compared in regard to the percentage of bees that responded in the test situation
• However, much more information can be obtained by videotape analyses of the response to an odor
• I have several examples now in which the percent response levels in two treatment groups are identical, but the groups differ in other aspects of the response topologies (e.g. duration and latency)
• For video analyses make sure that the recording time extends for about 20 sec after onset of odorant, because a response that begins during the odor presentatation can easily extend beyond the offset of odor
• always use extinction (odor only) trials for video recording
• that is, don’t record acquisition trials, because you can only score latency in those kinds of trials (the response is contaminated by the presence of food)
• You can score many response measures from slow frame advance mode on the videotape
• duration = time that the proboscis broke the mandible line (above) subtracted from the time that the line is again broken during retraction
• multiple extensions/retractions can occur during the 20 sec recording and they must be calculated individually, even though you may ultimately decide to add all of them together to obtain the total time the proboscis was extended during the trial
• latency = time that the first extension occurs minus the time the light (odorant) comes on
• glossal extensions can be counted and timed relative to the onset of odorant, the first extension or their own timing parameters

• The following links go to video sequences from extinction (odor only) trials
• note that in all three examples the bees respond with proboscis extension to odor, so theyall would have received the same visual score (a '+')
• nevertheless, they all differ in terms of latency, duration, and the number of extensions/retractions during a single 4 sec odor pulse
• in each frame, the camera is focused directly at the front 'face' of the bee
• the mandibles and proboscis are oriented toward the top of the frame
• the two antennae are also free to move
• Each video frame contains the frame code in the lower right portion of the frame
• the number to the left of the colon indicates the number of seconds into the tape at which the frame occurs
• the number to the right of the colon indicates the number of frames into that second
• given that there are 30 frames/sec, you can convert to absolute time by multiplying the latter number by 0.033 and adding it to the seconds
• The red light at the bottom of the frame indicates that the odor is being presented
• bees cannot see red, and the light is placed behind and below the bee
• when this light comes on (the first frame in each sequence) the time reference for the trial is set to 0
• Also, note the antennal movements
• this page focuses on proboscis extension as a measure of the conditioned response
• but the antennae also show characteristic odor-oriented movements that can be conditioned
• SHORT LATENCY/LONG DURATION
• LONG LATENCY/SHORT DURATION
• SHORT LATENCY/SHORT DURATION

• Educate yourself about Interstimulus Interval (ISI) and Intertrial Interval (ITI) by reading Rescorla (Ann. Rev. Neurosci., 1988)
• Describe conditioning and test trials
• use different (new/charged) syringes for testing
• the ones used for acquisition may be differentially depleted if you've used them for different numbers of trials

• Remember: bees like it hot! We keep our training rooms about 82-85^oF (29-30^oC)
• Contact Brian H. Smith (smith.210@osu.edu) for questions/problems

• I is VERY important to accuratly and precisely regulate the delivery of the CS and the US
• You will need a means to automate the odor delivery system
• we use our own hardward that is driven by a parallel port on a computer system
• The parallel port controls a series of valves that regulate the flow of air through the odor cartridges
• We use syringes that can delivery as little as 0.2 microliters of the sucrose/water USFnally, you'll need a good exhaust system to evacuate the odor-laden air from the conditioning arena
• If you would like more information about sources for these items, or if you would like to obtain a copy of our software, contact Brian H. Smith (smith.210@osu.edu)