During classical associative learning, an animal is taught to associate a neutral conditioned stimulus (CS) with an aversive unconditioned stimulus (US). Classical conditioning of the eye-blink response in rabbits uses the association of a neutral stimulus such as tone or light with a nociceptive stimulus, such as an airpuff delivered to the eye or a periorbital shock. Re-presentation of the CS results in an eye-blink conditioned response (CR) in anticipation of the US. Trace eye-blink conditioning (CS followed by an intervening time delay) is a hippocampus-dependent form of associative learning, while delay conditioning (no intervening time delay) is hippocampus-independent. An elegant series of studies has implicated a role for long-term depression (LTD) in the cerebellum in eye-blink conditioning. Additional sophisticated studies have also mapped much of the relevant neuronal circuitry underlying this behavior. We will return to these studies in more detail in later chapters, where we discuss the general role of synaptic plasticity in learning and memory.
Corneal Air Puff Elicits Eye-Blink Response
Corneal Air Puff Given with Tone
Tone Given Alone Elicits Eye-Blink Response
BOX 2 Eye-blink conditioning in rabbits. Animals are trained in a Pavlovian conditioning paradigm to learn that a tone predicts a puff of air to the eye surface. Over time, the animals learn to blink in response to the tone alone. See text for additional details.
and translation of genes downstream of the CyclicAMP Response Element (CRE) DNA regulatory element (see reference 3 and Figure 14). A variety of prior "block"-type studies in a variety of model systems suggested that the CRE transcriptional pathway was involved in long-term memory. However, no mammalian behavioral model system had yet demonstrated that environmental signals associated with learning led to alterations in CRE-mediated gene expression. Impey and Storm undertook a "measure" study to assess this issue directly. They found that training for contextual fear conditioning or passive avoidance led to significant increases in CRE-dependent gene expression in the hippocampus. Auditory-cue fear conditioning, which is amygdala-dependent, was associated with increased CRE-mediated gene expression in the amygdala but not in the hippocampus. These studies demonstrated that behavioral conditioning activates the CRE transcriptional pathway in specific areas of the brain associated with specific learning behaviors—a fine example of applying the "measure" experiment in a behavioral paradigm.
FIGURE 14 Contextual conditioning and CRE-mediated gene expression in mice transgenic for CRE-Lac Z. (A) Summary of associative learning (contextual fear conditioning) measured 8 hours after training. Mean percentage of time spent freezing in the conditioning chamber is depicted for naive, unpaired control and context-trained mice (naive, n = 7; unpaired, n = 8; context-trained, n = 23; naive versus context and unpaired versus context, p < .0005). (B) In these experiments hippocampal CRE-mediated gene expression was assessed using Lac Z expression (see text). (B) Low-magnification confocal images show CRE-regulated Lac Z immunostaining in hippocampal slices from representative naive, unpaired, control, and context-trained mice. Scale bar represents 500 mm. (C) Higher-magnification images of the CA1 region from representative naive, unpaired control and context-trained mice. Scale bar represents 100 pm. (D) Quantification of Lac Z immunostaining in area CA1 (unhandled control, n = 5; naive control, n = 8; unpaired control, n = 8; context-trained, n = 23; naive versus context and unpaired versus context, p < .001; p > .3 for comparisons between control groups). (E) Quantitative analysis of Lac Z immunostaining in area CA3 (unhandled control, n = 5; naive, n = 8; unpaired, n = 8; context trained, n = 12; naive versus context, p < .01; unpaired versus context, p < .02; p > .5 for comparisons between control groups). (F) Summary of Lac Z immunostaining in the dentate gyrus (control, n = 5; naive, n = 8; unpaired, n = 8; context trained, n = 11; naive versus context and unpaired versus context, p > .3). Figure adapted from reference 3, courtesy of Dan Storm.
I point out these studies in part because they are such an elegant combination of behavioral and transgenic animal approaches. However, I also like to use these studies to illustrate how assaying molecular changes using behavioral paradigms necessitates some rethinking of these paradigms because they have been historically developed using a behavioral perspective. One of the controls that is necessary in an experiment like the one described above is a foot shock alone control, where the animal experiences the foot shock under a condition where it does not associate the foot shock with a particular context. This allows you to demonstrate that the molecular change is not due to a direct sensory response to the shock, for example, but rather is selective for the condition where the animal is actually learning something. However, how do you keep the animal from learning to associate the foot shock with the place where it is shocked? One way to solve this problem is to repeatedly expose the animal to the place in which you will shock it, in the absence of foot shock. In other words, you habituate them to the shock environment prior to delivering the foot shock so that no unique association is made between the foot shock and that environment. Then you can deliver the shock alone in the absence of the animal learning an association. This procedure works quite well behaviorally, and the animal exhibits no fear conditioning to the shock environment. It also works in molecular studies like the one described earlier—no molecular changes in CRE-dependent gene expression occur in the hippocampus with an unpaired control like this.
However, consider what happens when you put a naive animal in the context and deliver a foot shock—conditioning and changes in CRE-dependent gene expression both occur. Thus, in the "control," habitua-tion to the context has made the hippocampus refractory to foot shock-induced molecular changes. This refractoriness of course must itself be mediated by some molecular change in the hippocampus (or its input pathways). Thus, the behaviorally straightforward "shock alone" control becomes the molecularly complicated "refractoriness to contextual learning" experimental sample. I want to make clear that this consideration in no way diminishes the legitimacy of the conclusions that Impey and Storm drew from their experiments. I use this simply as an example of the complexities involved in trying to transition from behavioral studies that use behavior as a read-out to behavioral studies that use cellular changes or molecules as a read-out.
We ran up against a similar puzzle in some experiments that Coleen Atkins and Joel Selcher did in my laboratory a few years ago (10). They were using contextual conditioning (context alone paired with foot shock) and contextual-plus-cued (context plus white noise auditory cue paired with foot shock) conditioning protocols and looked for changes in protein kinase activation in the hippocampus. They observed a significant activation of hip-pocampal Calcium/calmodulin-dependent Protein Kinase (CaMKII) that was selectively associated with the context-plus-cue conditioning paradigm (Figure 15). Why is there a unique hippocampal effect associated with the context-plus-cue paradigm versus the context alone paradigm? After all, in both paradigms the animal is learning to associate the identical context with the foot shock, and the cued component is not dependent on the hippocampus. We do not know the answer, but one interesting possibility is that the change is indicative of the animal having associated the noise cue with the context. Another possibility is that the CaMKII change is a manifestation of the animal having formed a unique multimodal association of cue, context, and foot shock. Again, I raise this point as an example of how we are likely to
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