The overall goal of this experiment is to demonstrate how the use trace eyeblink classical conditioning, a form of associative learning, can help detect hippocampal dysfunction in a rat model of fetal alcohol spectrum disorders. Neonatal rat pups are challenged with a high dose of alcohol, a concentration of 11.9%volume volume alcohol in a binge like fashion. During the equivalent of a third trimester in humans with respect to brain growth.

This pattern of alcohol exposure and dose are key factors in producing brain deficits over the life span mimicking human cases of FASD's. An important brain region that is compromised by early alcohol exposure is the hippocampus. After the rats grow up as adults, they are examined for hippocampal dysfunction using trace eyeblink classical conditioning.

This is a high order form of associative learning that relies on the integrity of the hippocampus. During post natal days four through nine, rat pups are weighed each morning and their weights are recorded. Feeding volumes are then obtained from a pre printed table based on body weight.

An 11.9%volume volume alcohol solution is delivered to rat pups over two feedings that are separated by two hours. The total daily dose of alcohol is 5.25 grams per kilogram per day. This binge exposure paradigm delivers 0278 mils of alcohol per gram body weight per feeding in milk solution.

Extract the milk containing alcohol using a sterile one milliliter syringe. Then push out excess solution to obtain the exact amount needed. Dip the tip of the PE-10 tube in fresh corn oil.

This facilitates insertion. Measure the length of the PE-10 tube from the pup's mouth to its stomach. If necessary, adjust the PE-50 stopper to act as a visual guide for the stopping point.

Carefully insert the PE-10 tube into the pup's mouth. You will typically encounter some resistance as the tip meets the throat. Maneuver the tube until the tip bends into the entry of the esophagus.

Try not to let the tube slip backwards, as it may be slippery with the oil. Proceed down its esophagus and as the tip passes the gastroesophageal sphincter, it will then enter its stomach. Stop at this point, stabilize the pump, and deliver the solution.

This should be done at a slow rate. Carefully remove the PE-10 tubing and examine the pup for backwash of solution, blood, or physical injury. Replace pup with its litter mates if it is fine.

Refer to the written protocol and materials list for specific instruments used in the eyelid surgery procedure. Sterilize the surgical field with an approved disinfectant and autoclave all surgical instruments and supplies ahead of time. After inducing proper anesthesia in the rat with isoflurane, shave its head using a fur trimmer, exposing a sufficient amount of skin for the incision site and the left eyelid.

Its skin is sterilized by performing three alternating scrubs involving 70%isopropyl alcohol and povidone iodine. Follow standard procedure in securing the rat to the stereotaxic device with the ear bars. Proceed with the surgery when the rat exhibits proper anesthetic plane.

Use the scalpel blade to make an anterior posterior incision at the midline. This incision should expose enough area anterior to the eyes and slightly posterior to the lambda sutoral line. Use the scalpel blade to scrape away the periosteum on top of the cranium, carefully, not to cause excessive bleeding.

Wipe off excess connective tissue and blood with a cotton tipped swab and rinse the area with sterile saline between scrapes. Drill a hole starting directly behind the coronal suture on one parietal bone. Remove any blood and bone debris with a cotton tipped swab.

Use splinter forceps to grasp a 0 80 screw. Use a jeweler screwdriver to fasten the screw to the hole, tighten down the screw just enough without damaging cortical brain tissue, usually three to four full turns. Follow the same procedure to secure the remaining screws.

There should be two screws secured bilaterally behind the coronal suture and one on the right parietal bone, anterior to the lambda suture. Grasp the upper skin at the incision site of its left eye with the four inch dressing forceps and direct the three inch dressing forceps towards the corner of this eye. Take one 26 gauge 3/8 inch needle and insert it though the corner of the eyelid.

Rotate the needle so that the beveled side is face up. Insert the second needle in the middle of the eyelid. Use fine forceps to grasp a 3T wire and insert it into one of the needles.

You do not need to insert it all the way. Follow the same procedure for the second 3T wire. Grasp both needles with one hand while grasping the head stage with the other hand.

In one continuous movement, pull the needles away from the eyelid while guiding the head stage in the same direction. While holding the EMG head stage with one hand, use the three inch dressing forceps with the other hand to wrap the 10T wire around one or both screws on the right side. Tuck away this wire so that it does not protrude from the screws.

The EMG head stage should be centered before moving on to the next step. Use the four inch dressing forceps to create a small pocket by separating the skin, that is, the superficial fascia from the temporalis muscle. With the pocket created, use the iris scissors to cut away more connective tissue, working in towards the corner of the left eye.

Be careful not to go in too deep or cut any blood vessels. Take a bipolar electrode and shape the wire lead so that they can be fitted along the curvature of the temporalis muscle. The two prongs will be situated posterior to the left eye and the bottom end of the bipolar electrode will be situated straight atop the cranium.

Mix the Ortho Jet powder in liquid components to obtain a liquid consistency. Apply the paste with a micro spatula all around the incision site. Be careful not to drop any paste on the rat's eyes, and keep it contained.

Allow the Ortho Jet to get to a semi dry state, then use the splinter forceps to remove excess acrylic on the EMG head stage. And the threads of the bipolar electrode. Snip away excess 3T wire, leaving a few centimeters to work with.

Carefully remove the PTFE shielding from each 3T wire using the micro dissecting forceps. This allows them to make contact with the orbicularis oculi muscle. Use the micro dissecting forceps to create hooks on each end of the 3T wires.

Snip away excess wire, being careful not to cut away so much that the remaining wire retracts back into the eyelid. The cabling is made in house. The white bipolar plug is connected first, and this allows delivery of the shock US stimulus.

The orange EMG head stage plug is connected afterwards. It records electro myographic activity as the rat blinks. Both plugs lead to a round commutator at the top of the arm which maintains signal continuity as the rat moves about freely.

The stimulus isolator is turned on and final hardware checks are made. This includes adjusting the EMG baseline to a level that is viewable on the computer screen. In session one, the shock intensity is first set to 4 milli amps and will be increased in 4 mil amp units every two trials.

This phasing in procedure allows a rat to acclimate to the shock stimulus. By the eighth trial, the shock intensity is maintained at 1.6 milli amps throughout this session and all subsequent sessions. A separate computer runs a four channel digital oscilloscope which allows us to perform real time signal diagnostics.

Note the eyeblink that was emitted by the rat in box one. The oscilloscope also indicates that the baseline EMG signal is relatively clean with low noise. Webcams allow us to monitor each rat as well.

This is a screenshot from blink software version 4.0 that shows a trial epoch lasting 1400 milliseconds. In the first 280 milliseconds, no stimuli are presented to obtain a measure of baseline EMG activity. The yellow line indicates the threshold line in which any EMG response that exceeds 4 volts above the average pre-CS baseline value will be eligible as an eyeblink response.

The tone condition stimulus of 85 decibels is delivered at the 280 millisecond time point. The tone remains on for 380 milliseconds which corresponds to the 660 millisecond time point. After it shuts off, there's a 500 millisecond interval where no stimuli are presented This is called the trace period where the rat must resolve the offset of the tone CS with the onset of the shock US, still to be delivered at the 1160 millisecond mark.

The shock US lasts for 100 milliseconds. Naturally, the shock US elicits a defensive blink response known as the unconditioned response, or UR that is measured in a 140 millisecond time window. One session of training consists of 100 trials.

There are 90 trials in which the CS and US are paired and on every tenth trial, only the CS is delivered. There are 10 CS only trials per session. These trials allow us to probe for whether or not learning of the CS US association.

In the form of condition responses, or CR's is expressed when the shock US is not present. The inter trial interval is averaged out to be 30 seconds, so that the start of each trial is not predictable. In any given trial, a rat may blink at any time point and its EMG will be recorded by the software.

Any discreet eyeblink that exceeds the threshold line will be considered a response in one of four categories based on when they occur and the trial epoch. If the rat blinks during the first 80 milliseconds after the tone CS onset, the response is considered a startle response or SR.This is a non associative response that is indicative of sensory motor performance, but not learning of the CS US association per se. If it blinks after the SR period but before the shock US is delivered, then it has emitted a conditioned response.

As mentioned previously, the CR is the primary measure of associative learning. The CR is not expected to develop during the early phases of training, but as training progresses over many trials and sessions, then these types of responses become more frequent, higher in amplitude, and may be more well timed. We partition CRs into two types, total CRs and adaptive CRs.

Total CRs reflect general learn ability while adaptive Crs reflect the animal's ability to time its blink well. Immediately before the onset of the shock, as a way to brace itself, the adaptive CR period is 200 milliseconds before shock US onset. In mammals with an intact hippocampus, the expression of adaptive CRs reflects high level hippocampal involvement in timing the blink properly in relation to delivery of the two stimuli.

By its very nature, trace eyeblink classical conditioning is much harder to acquire than delay eyeblink classical conditioning where the tone CS and shock US overlap in time. While the expression of CRs may vary from subject to subject and can be severely affected by challenges to discreet brain circuits, the UR is expected to be emitted during every paired CS US trial. As such, this measure reflects sensory motor performance and like startle responses, are not indicative of learning of the CS US association per se.

In the following series of video clips, you will see the software set up and the start of eyeblink training. The blink software is activated and the two pertinent files are recruited. A rat file containing subject data, and the trace eyeblink conditioning file.

The program is set in place until training begins. As training starts, notice that there's a timer at the right bottom of the screen that counts down to the next trial. Here is a live video recording of session one, trial 15, where a UR is emitted but no CRs have developed yet.

Here is a replay at one third of the speed. In the following series of clips, you will see screenshots from the same un intubated control rat as it progresses through six sessions of training. It eventually emits both total CRs and adaptive CRs.

Here is a live video clip of session six, trial 96, where two eyeblinks are emitted, one an adaptive CR, and the other a UR.Here is a replay at one third of the speed. In this final clip, you will see a live video of an alcohol intubated rat on its last trial of session six, a CS only trial, even after 600 trials of training, it doesn't emit an adaptive CR reliably. After the data are collected, they are screened for bad trials, consisting of high noise artifacts, and unusual pre-CS baseline EMG activity using a built in error algorithm for detecting outliers.

Good trials that are kept are further analyzed for SRs, URs, total CRs, and adaptive CRs with respect to percentage of responses, amplitude, latency, and other critical measures. Mean adaptive CRs across six sessions of training are shown in this first set of results. Both the percentage and amplitude measures were statistically significant between the alcohol intubated group and the two control groups from session two through session six.

This indicates that the frequency and the strength of adaptive CRs were impaired in rats exposed to alcohol during early development. Mean unconditioned responses are shown in this second set of results. Both the frequency and amplitude measures were not statistically different among any of the groups.

These results indicate that early alcohol exposure did not impair the rats'ability to blink to the unconditioned stimulus, but that the impairment they exhibited was due to a failure in associating the CS and US properly during acquisition. In this experiment, we examined the brain behavior relationship by determining whether or not developmental alcohol exposure that is delivered in a binge like fashion affects the hippocampus as reflected in deficits and trace eyeblink classical conditioning. Indeed eyeblink classical conditioning is a useful tool to assess the neurobehavioral consequences associated with alcohol exposure during brain development.

It's very versatile in that by changing the stimulus parameters or associative learning requirements the functional integrity of different brain systems outside the hippocampus can also be assessed. After watching this video, you should have a good understanding of the intricacies involved with delivering alcohol to neonatal rat pups, implanting electrodes, and carrying out the eyeblink testing procedure. We thank you for watching this video.

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