Measuring CTA Expression
When a taste is paired with a suspected toxin, the animal may (or may not) learn a Conditioned Taste Aversion. The only way to tell if the animal has a CTA is to try to measure the CTA in an expression test (also called a retrieval test.) An expression test is also called an extinction test, because the taste is presented without the toxin: if the taste is repeatedly presented without the toxin, the animal will “unlearn” the association of taste and toxin and the CTA will be extinguished.
There are several ways to measure CTA expression. They vary in the aspect of CTA learning that is measured, and in their sensitivity.
Taste reactivity (TR)
One of the features of CTA is that it can change the reflexive response of the animal to a taste, i.e., the unconditioned rat responds to the taste of sucrose with ingestive orofacial movements, such as licking, tongue protrusions, and swallowing. After the rat acquires a CTA, however, it now reverses its reflexive response: it vigourously spits out the sucrose, shakes its head, and “gapes” – the rodent equivalent to vomiting (which they are physically incapable of doing). This change in orofacial responses can be quantified by implanting an intraoral catheter into the mouth of the rat, and videotaping the rats head as the conditioned taste is infused directly into the mouth for a brief period (30s). The behaviors of the rat are scored from the videotape frame-by-frame. If the rat has a CTA, the number of ingestive responses will decrease and the number of aversive responses increases.
The advantage of TR is that it is very precise both in terms of the stimlus presentation and the response measurement: it measures the rats direct orofacial motor response to a fixed taste stimulus through the catheter, and it is very quantitative – the stronger the aversion, the more aversive responses. The disadvantages are that it is very labor intensive, it is not a common measure (so the historical database is not large), and it is a complicated and unnatural method of measuring a rat’s response to a taste (so there is a greater contextual influence on the learning.) Furthermore, intraoral techniques are not very sensitive: only relative strong CTAs will affect TR strongly.
Intraoral (IO) Intake
IO intake tests use the same set-up as TR: the rat is implanted with an intraoral catheter, and the conditioned taste is infused directly into the mouth. The rat can swallow the solution, let it drip passively from the mouth, or actively spit it out. To measure intake (instead of just orofacial responses) the infusion continues for 5-30 min. If you weigh the rat before and after the infusion, you get a measure of intake (whatever the rat swallows, it gains as weight). The IO intake measure has many of the advantages and disadvantages of TR: it is labor intensive, but the data collection is easier (weight gain instead of scoring videotape), and the measure of intake is the same measure as used in traditional bottle tests. As with TR, IO intake seems less sensitive to weak CTAs.(You can imagine situations in which a rat might not voluntarily drink a solution, but if forced to taste the solution, it decides its not so bad and starts swallowing it….)
Single bottle test
A one-bottle test is a forced choice test of CTA expression. Typically, the rat is maintained on a water deprivation schedule, in which it has access to a single water bottle for only 10 min – 6 h each day. under these schedules, the rat is usually thirsty and will drink from the bottle as soon as it is presented. On the test day, the rat is presented with a single bottle of the test solution instead of water. If the rat has a CTA, it will drink less of the conditioned taste solution than a control rat. A single bottle test is more sensitive than IO measures (i.e. the rat will show a significant aversion with weaker CTAs (such as after a pairing with a lower dose of Li) than will be seen intraorally.) However, there is the possibility that the rat is too thirsty to avoid drinking altogether. Because the test solution is its only source of fluid, it may drink even if it has a CTA. The briefer the single bottle test, and the less water-deprived the rat, the greater the sensitivity of the single-bottle test. Furthermore, the single bottle test is very easy to carry out, and very precise measures can be made if the rat’s licking at the bottle is recorded by a computerized lickometer.
Two-Bottle test
A 2-bottle test is a choice test of CTA expression, and is very sensitive. Typically, a water deprived rat is given 2 bottles to drink from: one with the conditioned taste solution, and the other with water or a neutral solution (a taste never paired with the toxin – also called a CS-). This is a very sensitive test, because the thirsty rat does not have to drink the test solution to rehydrate at all – it can drink from the other bottle. It is a little more effort than a single bottle test, however, to measure the intake from 2 bottles. And you have to make sure the rat knows that it can drink from either one of 2 bottles (so in the days leading up to the test, you should give the rat a full water bottle and an empty water bottle, alternately left and right positions on the cage, so it knows to try either drinking spout.)
As with a single bottle test, the short-term measure (10 – 60 min) is usually more sensitive than a long-term measure (24 h), because the rat may be extinguished when it has long-term access to the solution without any pairing of the taste with the toxin.
There are 3 ways to examine the results of these intake tests:
- If you give the rat a CTA by multiple pairings of the taste and toxin, you can measure the rate of acquisition. Does it take 1 pairing, 2 pairings, or 3 pairings of taste and toxin to get a CTA of a certain strength? How much does the intake of the conditioned taste solution start to go down during the acquisition trials (i.e. during the consecutive pairings.)
- After the pairings of the taste and toxin, the CTA can be measured by the intake or TR response on the first expression test.
- It is possible that on the first day of expression testing, all strong CTAs will suppress intake completely or to the same level and so be indistinquishable on the first day (i.e. a floor effect). By measuring the rate of extinction across repeated, unpaired expression tests, the strength of CTAs can be distinguished even if they appear equally strong on the first day of testing. Weaker CTAs will extinguish faster than stronger CTAs, so the number of extinction trials it takes to reach control levels of intake will be proportional (in some sense) to the strength of the CTA.
One of the goals of our laboratory is determine the algebra by which underlying quantifiable molecular events result in quantifiable behavioral expression of the CTA.