Pseudo-conditioning

One of the main pitfalls researchers need to guard against, when attempting to identify conditioning in animals, is pseudo-conditioning. Pseudo-conditioning refers to behaviour mimicking conditioning, whereby "presentation of the US can affect the animal so that a subsequently presented CS produces a 'CR' [conditioned response - V. T.], even though the CS and US have never been paired" (Abramson, p. 156). Likewise, in instrumental and operant conditioning trials, the reinforcement alone may produce a behavioural change resembling the change which is supposed to be produced by conditioning. Abramson gives an example of a crab which is trained to press a lever for food, by being rewarded with squid after each bar press. After the first few bar presses, the rate of pressing increases dramatically. Is this because the crab has associated the lever with the reward (a mentalistic interpretation) or because of the energizing effect of the food, independent of any link between response and reward? According to Abramson (1994, pp. 129, 156), pseudo-conditioning can be excluded by training the animal to discriminate between two stimuli, where only one is paired with the US (in classical conditioning experiments) or where only one leads to the reward (in the case of instrumental conditioning). For instance, if the crab in the above experiment were able to discriminate between two levers, only one of which is reinforced by food, then conditioning would be demonstrated.

Sensitization

Another explanation for apparent "conditioning" in animals is sensitization. What is supposed to happen in classical conditioning is that the animal learns to associate a neutral event (the conditioned stimulus) with a biologically significant non-neutral event (the unconditioned stimulus). However, Abramson cautions that often, researchers working with invertebrates end up using a conditioned stimulus which is non-neutral, as it "already elicits the conditioned response, albeit in a reduced form" (personal e-mail, 2 February 2003). This is a pervasive problem with studies of invertebrate learning:

Unfortunately, most of the CSs used in invertebrate behavioral experiments elicit the response that is to serve as the index of learning (Abramson, 1994, p. 133).

If the index of "learning" is the same as one of the responses elicited by the CS prior to pairing, then measurement of learning becomes problematic. Abramson discusses this issue in connection with planarians (a kind of flatworm):

The planarian work revolved around the same issues. It is difficult to find a CS that does not produce a response resembling a conditioned response prior to pairing (there is only so much variety in the behaviour of worms) (personal email, 2 February 2003).

Alpha conditioning

Some researchers have attempted to circumvent this problem by habituating the animal to the CS before conditioning trials begin, in order to make it seem benign or "neutral" to the animal. When the animal ceases to respond to the CS, the CS is then paired with the US, until the animal responds to the CS in the same way that it (innately) responds to the US. The problem with this procedure is that the new response elicited by the CS may simply be due to a re-awakening of the animal's old, innate response to the CS (by pairing it with the US, which evokes a similar response), rather than a learned association between the CS and the US. On the former explanation, the animal's behaviour can be explained as a kind of sensitization known in the literature as conditioned sensitization. Other authorities refer to it as alpha conditioning or US-US conditioning, as the CS elicits an innate response and should therefore be described as a (non-neutral) US rather than as a (neutral) CS (Abramson, 1994, p. 106). The difference between the two explanations is that in the former case, the animal is not learning to do something new; it is merely being habituated to a stimulus and then re-sensitized to it. As Abramson puts it:

If we go back to the planarian work, standard procedure was to first HABITUATE responses to the CS prior to pairing with the US. In my view this experimental design is not classical conditioning... After the first presentation of the US the animal is now sensitized once again... If you were to plot the performance (conditioned response) of such a procedure, the first CS trial begins at '0' and the reader has the impression that the CS was neutral (personal e-mail, 2 February 2003).

Other factors

Other factors that may lead to false positive results in conditioning trials are: (i) the base rate of responding (where the animal shows a tendency to respond spontaneously, without any stimulation); (ii) the animal's central excitatory state or CES (where its nervous system may be excited by the presentation of the unconditioned stimulus, so that it responds to stimuli which it would not normally respond to - including the conditioned stimulus); (iii) pheromones, or signalling odors, which may contaminate the experimental apparatus and alter the animal's behaviour on subsequent trials); (iv) calendar variables (e.g. seasonal or local weather changes) which affect animals' behaviour; and (v) experimenter bias (e.g. subtle differences between researchers in the way they present stimuli to the animal) (Abramson, 1994, pp. 130-132, 157-159). Abramson points out that contamination of results by these factors can be avoided by using suitable control groups, longer inter-trial intervals, proper cleaning procedures and "blind" experiments (to rule out bias). However, the problem is that conditioning trials for invertebrates are seldom conducted rigorously enough to rule out these factors.

Pitfalls in experiments with flatworms

Claims by scientists to have identified classical conditioning in planaria (flatworms) have been discredited by subsequent research. The following account illustrates how sloppy experimental controls can lead researchers to draw premature conclusions about animals' capacities for classical conditioning:

Classical conditioning of planaria was first reported by R. Thompson and J. V. McConnell in 1955. The planaria became a fairly common model for studying conditioning and memory in the 1960's. In 1967, Richard Block and McConnell published an article in Nature, that reported results of classical conditioning in brown planaria (Dugesia dorotocephala). In their study, they paired an electrical shock ... with a flash of light ... When the shock would occur, the worms would contract and turn at the anterior end. The experiment was controlled by a group that received no electric shock, and by a group that received light and shock at random. After training, the planaria were given stimulus by light alone without electric shock. The planaria would exhibit the same type of response that was originally caused by the electric shock. Block and McConnell followed this with extinction trials, that is they "reverse trained" the planaria to forget their earlier learning.

Studies of this kind fell out of favor in the early 1970's, as some researchers, notably Allan Jacobson, Sheldon Horowitz, Clifford Fried, John Hullett, and M.J. Homzie, argued that the training was not true classical conditioning, but rather that the planaria exhibited "pseudo-conditioning" and "sensitization" (Duane, Fortenberry and Horowitz, 1998).

Pitfalls in experiments with earthworms

In a similar vein, Abramson and Buckbee (1995) report on four experiments they conducted with earthworms (Lumbricus terrestris). Earthworms represent what Abramson (1994, p. 177) calls "the next evolutionary advance" beyond flatworms, in that their body is divided into similar segments, which "can respond individually or as a group, and can be modified to perform special tasks" (1994, pp. 177-178). By using multiple control procedures, Abramson and Buckbee demonstrate that much of what has been described as "associative learning" in earthworms is actually non-associative in nature. Earthworms were trained to respond to the scent of rose (the conditioned stimulus) in the same way as they responded to the smell of n-butanol (the unconditioned stimulus, to which earthworms have an innate aversive response). The fact that earthworms who were exposed to the US before being exposed to the CS (backward conditioning) responded no differently from earthworms who were exposed to the CS before the US, tells against the hypothesis that they had learned to associate the CS with the US, and in favour of the hypothesis that their response was due to sensitization. Previous experiments have tended to overlook backward conditioning; instead the control group used has been one where the CS and US are simply not paired. Abramson and Buckbee suggest that "relying solely on an unpaired group may underestimate the amount of pseudo-conditioning" (1995, p. 394). They also note that they "were surprised to find no studies in the worm literature directly measuring the extent of pseudo-conditioning" (1995, p. 394).

Abramson and Buckbee (1995, p. 395) also describe an experiment in which they attempted to measure the extent to which earthworms' responses to the scent of the CS were caused not by an association between the CS and the US, but by a carry-over excitation of their nervous systems arising from the presentation of the US during a previous trial, making them respond to stimuli which they would not normally respond to - including the CS. What passes for a conditioned response may in fact be due to the central excitatory state (CES) of the subjects. In their experiment, Abramson and Buckbee conditioned the worms in 20 trials where the CS was immediately followed by the US, and then (one minute later) by a third stimulus that was supposed to dissipate the lingering effects of the US, before the next trial. Trials were spaced at 2-minute intervals. The worms were equally likely to respond to the third stimulus (which did not predict the presence of the US) as to the CS, suggesting that the third stimulus "did not effectively discharge unconditioned stimulus excitation or contained carry-over excitation of its own" (1995, p. 395).

Summing up, the authors conclude that the animals failed to display true classical conditioning, and that their apparent "learning" responses "can be interpreted as an example of sensitization as measured by either a pseudo-conditioning or by a CES design" (1995, p. 395). They suggest that "sensitization appears to be the underlying behavioural mechanism of what has been previously characterized in the earthworm as classical conditioning phenomena" (1995, p. 396).

An over-drawn contrast?

Finally, the contrast which is often drawn between two forms of "learning" - non-associative (where the animal modifies its response to one kind of event but does not learn to do anything new) and associative (where the animal learns to associate two kinds of events) - may be an over-simplification. Work by Rose and Rankin (2001) suggests that associative cues can facilitate habituation: C. elegans worms remember their habituation better if they are tested in the same environment they were trained in.