Classical Short-Delay Eyeblink Conditioning in One-Year-Old Children

The overall goal of this procedure is to assess associative learning in one-year-old children using eyeblink conditioning. The eyeblink conditioning paradigm is useful for detecting certain types of learning and memory deficits. For example, adults and children with fetal alcohol spectrum disorder have deficits in eyeblink conditioning.

This is due to disruptions to their cerebellar circuitry. Eyeblink conditioning refers to a learned association between an auditory tone and a puff of air to the cornea that stimulates an eyeblink. With repeated pairing of the two stimuli, children may learn to blink when they hear the tone, in anticipation of the puff.

This method can be used to investigate neurodevelopmental deficits. Although challenging to administer in young children, the earlier we can detect and treat these children, the more likely they are to achieve the best possible outcome. eyeblink conditioning units are commercially available, or can be custom-made.

The set-up will require the following equipment:an air puff unit capable of administering a puff of air directly to the cornea, using an air compressor with pressure regulator. This should be attached to an adjustable headband or hat that can be worn by the infant. Two portable audio speakers with adjustable volume settings.

A video camera with at least a 60-hertz frame rate to capture the blink responses. Computational control of the equipment, so that the tone and air puff can be administered automatically with precise timing and at varying intervals. The eyeblink conditioning stimulus described here consists of a 750-millisecond tone, which overlaps and terminates with a 100-millisecond air puff.

To examine changes in associative learning over time, deliver the trials in blocks of 10, with a total of 50 trials. In each block, include one tone trial without the air puff. This will allow you to directly assess for associative learning.

In each block, also include one air puff trial without the tone. This will allow you to confirm the air puff unit is correctly positioned and that the child is sensitive to the air puff. Place two portable speakers on either side of the infant.

Position the infant on the caregiver's lap. While one researcher distracts the infant, a second researcher can place the headband on the infant's head and adjust for size. Position the flexible tube holding the air puff nozzle and infrared sensor so that these are approximately one to three centimeters from the child's eye.

Before beginning the experiment, check that the air puff unit is correctly positioned. Do this by delivering a single test air puff to the infant's eye and observe whether an eyeblink occurs. Some repositioning of the headband may be necessary.

Automated eyeblink detection methods included with commercial setups are often unreliable in young infants as it is difficult to maintain the sensor close enough to the eye to detect the eyeblink. Instead, frame by frame video camera analysis should be used to detect eyeblink responses from infants. To do this, use a handheld video camera to manually track the infant's gaze during the experiment.

Frame by frame analysis of video recordings can be easily achieved using simple video-editing software. Detect the onset of the tone or the air puff using the audio waveform. To record the blink onset, look for the first frame where the eyes begin to close.

Researchers may also wish to note the peak of the blink, where the eyes are maximally closed. Calculate the number of intermediary frames and convert this response time into milliseconds based on your camera's frame rate. Conditioned responses are blinks that begin before the air puff is delivered.

Unconditioned responses are reflexive blinks to the air puff that occur more than 650 milliseconds after the tone onset. Other types of responses will also be observed. For example, startle responses are blinks that occur within the first 200-milisecond interval after the tone and represents a reflex response to the auditory tone or blinks timed coincidentally with the stimulus.

Finally, failed responses are trials where no blink was detected in response to the air puff. These may indicate that the air puff was not reaching the eye. Although frame by frame video analysis requires some subjective interpretation, this method can be used to reliably measure eyeblink latency from the stimulus onset.

Our comparison of 94 blink responses shows very high agreement between two observers which fall within a 250-millisecond limit of agreement. This variability is sufficiently low to distinguish conditioned from unconditioned responses. Responses that differ significantly should be reassessed by both observers.

Responses are likely to be highly variable between infants. Children who've achieved conditioning will respond before the air puff is delivered. They also may blink when the tone is presented without the air puff, shown by the red squares.

However, some infants show no evidence of conditioning during the experiment. Here the eyeblink responses occur after the air puff onset, suggesting that these are reflexive blinks. In addition, when the tone is delivered in isolation, the infant does not anticipate the air puff delivery.

Conditioning is best described as the change in number of conditioned responses over multiple trials or multiple experimental sessions. The most important step in obtaining reliable results is to maintain correct positioning of the air puff unit throughout the experiment. The infant should blink in respond to most air puff trials.

In this example, blink responses to the air puff decrease over the course of the experiment indicating that the air puff unit was not maintained in the correct position. The infant will not achieve eyeblink conditioning if they cannot sense the corneal air puff. Eyeblink conditioning is a useful paradigm to assess neural development in children.

What we've found is that video recording and subsequent offline analysis is a more reliable method when using this technique in young children.