A developmental dissociation in reinstatement of an extinguished fear response in rats

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Abstract

Recently, studies from our laboratory have shown that 16-day-old rats, in contrast to 23-day-old rats, fail to show either ABA renewal or recovery of an extinguished fear response following a pre-test injection of FG7142 [Kim, J. H. & Richardson, R. (2007). A developmental dissociation of context and GABA effects on extinguished fear in rats. Behavioral Neuroscience; Yap & Richardson, unpublished data]. The present study, using freezing as a measure of learned fear, extends these findings by examining whether there is a developmental difference in susceptibility to reinstatement following extinction. 16- and 23-day-old rats were trained to fear a white-noise conditioned stimulus (CS) by pairing it with a shock unconditioned stimulus (US). This fear was subsequently extinguished by non-reinforced presentations of the CS. Some rats received a post-extinction Reminder which consisted of a single presentation of a reduced-intensity US. Experiments 1 and 2 demonstrated that this Reminder was effective in reinstating extinguished fear in 23-day-olds, and that this reinstatement effect was context-specific in rats this age. In contrast, 16-day-old rats failed to show the reinstatement effect in either experiment. The failure to observe a post-extinction reinstatement effect in the 16-day-olds was not due to a general ineffectiveness of the Reminder treatment at this age because it did alleviate spontaneous forgetting in rats this age (Experiment 3). Taken together, the results suggest that fundamentally different processes may mediate extinction early in development compared to later in development.

Introduction

Learned fear is typically acquired by pairing an initially neutral conditioned stimulus (CS; e.g., tone) with an aversive unconditioned stimulus (US; e.g., footshock). The formation of an association between the CS and the US is inferred when subsequent presentations of the CS elicits various conditioned fear responses (CR; e.g., freezing). These learned fear responses can be reduced, however, by giving non-reinforced presentations of the CS—a process referred to as extinction. Extinction has received considerable attention over the past decade, partly because of its theoretical importance and partly because understanding the processes by which fear is diminished is critically important to the development of effective treatments for various anxiety disorders (for review, see Davis & Myers, 2002). Nevertheless, there is still relatively little known about the mechanisms mediating extinction, especially in comparison to what is known about the mechanisms mediating acquisition of learned fear.

Early models of associative learning suggested that extinction was due to the ‘unlearning’ or ‘erasure’ of the original CS–US association (e.g., Rescorla & Wagner, 1972). However, it is now widely accepted that the decrease in the CR following extinction reflects new learning of a second, competing association that inhibits the expression of the original association (i.e., CS–noUS vs. CS–US; for review, see Bouton, 2002). The primary evidence for this view comes from behavioral studies where performance to an extinguished CS recovers without subsequent re-training of the CS–US association. For example, responding to an extinguished CS can return over time, a phenomenon referred to as spontaneous recovery. Further, renewal refers to the recovery of an extinguished CR when the subject is tested in a different context to where extinction occurred. These findings indicate that the decrease in the CR after extinction training does not reflect an erasure of the original association, but is instead due to the suppression or inhibition of the original association by a competing association acquired during the extinction session.

Another finding that suggests that extinction is due to new inhibitory learning that masks the original excitatory association is reinstatement, which is the recovery of an extinguished CR following a post-extinction presentation of a ‘Reminder’ cue (Delamater, 1996, Pavlov, 1927, Rescorla and Heth, 1975). Typically, the reminder cue involves a re-exposure to the US alone (often of a slightly lower intensity than at training) or some other stressful experience. Although reinstatement is studied extensively in the laboratory, not much is known about the mechanisms underlying this phenomenon. However, reinstatement does appear to be context-dependent; that is, post-extinction reinstatement is often restricted to the context where the Reminder had been presented (Bouton, 2002, Bouton and Bolles, 1979, Ledgerwood et al., 2004; but see also Richardson, Duffield, Bailey, & Westbrook, 1999; Westbrook, Iordanova, McNally, Richardson, & Harris, 2002). From this, it would appear that a learned association between the context and the post-extinction US Reminder disinhibits responding to the extinguished CS. Consistent with this view, Bouton and his colleagues reported that rats failed to show reinstatement following lesions of the hippocampus (Frohardt et al., 2000, Wilson et al., 1995), a structure critically involved in contextual learning (for review, see Anagnostaras, Gale, & Fanselow, 2001).

It is also the case that a functional hippocampus is required in order to observe the renewal of an extinguished CR (Corcoran and Maren, 2001, Corcoran and Maren, 2004, Ji and Maren, 2005). The apparent requirement that the hippocampus be functionally active in order for either the reinstatement or renewal effect to be observed in adult rats leads to some interesting hypotheses regarding extinction in the developing rat. Because hippocampus is a late-maturing structure (Wilson, 1984), and it has been observed that young rats are impaired in learning about context (Pugh and Rudy, 1996, Rudy, 1993, Rudy and Morledge, 1994), one would predict that young rats would not exhibit either the renewal or the reinstatement of an extinguished CR. However, we know very little about extinction during development. Examining these extinction-related phenomena in the developing animal not only provides a unique way of assessing the processes involved in extinction, but is also important because of the long- and widely-held belief that early learning experiences have a profound impact on later behavior (e.g., Harlow, 1959, Mineka and Zinbarg, 2006). Jacobs and Nadel, 1985, Jacobs and Nadel, 1999, for example, suggested that fear acquired early in development is particularly resistant to the effects of extinction, and forms the basis of anxiety disorders emerging later on in life. However, there is very little, if any, empirical data to support this assertion. In fact, in one of the few systematic studies on extinction across age, Campbell and Campbell (1962) trained 25-, 50-, and 100-day-old rats on an avoidance procedure. When tested across 4 days, rats at all three ages exhibited comparable rates of extinction. That study and others that followed (e.g., Nair et al., 2001, Richardson et al., 2002), however, did not examine extinction-related phenomena like renewal or reinstatement.

We have begun to systematically examine several extinction-related phenomena in the developing rat. For example, several recent studies from our laboratory have shown that post-natal day (PND) 16 rats fail to show ABA renewal whereas PND 23 rats do show this type of renewal (Kim and Richardson, 2007, Yap and Richardson, unpublished data). The failure to observe renewal in PND 16 rats in these studies was not due to spontaneous forgetting, as non-extinguished controls displayed robust freezing at test. These findings suggest that a fundamentally different process may mediate extinction early in development. Therefore, the present study aimed to extend these findings by examining whether there is a developmental difference in susceptibility to reinstatement following extinction of learned fear. Specifically, we examined whether PND 16 rats were less likely to exhibit a return of fear following a post-extinction US Reminder than were PND 23 rats. Finding a developmental difference in the occurrence of post-extinction reinstatement would provide further evidence that a fundamentally different process may mediate extinction early in development.

Section snippets

Subjects

All experiments used experimentally naive Sprague–Dawley derived rats, bred and housed in the School of Psychology, University of New South Wales. Rats were either PND 16 or 23 at the start of an experiment. All rats were male, and no more than one rat per litter was used per group. Rats were housed with their littermates and mother in plastic boxes (24.5 cm long × 37 cm wide × 27 cm high) covered by a wire lid. Animals were maintained on a 12 hr light/dark cycle (lights on at 6am) with food and water

Experiment 1

We recently reported that PND 16 rats fail to show renewal of an extinguished fear response whereas PND 23 rats do exhibit renewal (Kim and Richardson, 2007, Yap and Richardson, unpublished data). The failure to observe renewal in PND 16 rats indicates that the contextual modulation of extinction may be impaired in rats this age. Experiment 1 aimed to extend these findings by examining post-extinction reinstatement, which also appears to be contextually mediated. We predicted that PND 23 rats,

Experiment 2

We observed reinstatement of an extinguished fear response following a post-extinction US in PND 23 rats but not in PND 16 rats in Experiment 1. In Experiment 2 we attempted to replicate this finding, and to also examine the context-specificity of post-extinction reinstatement. At each age some rats were given the reinstatement treatment in the same context in which they would be subsequently tested while other rats were given the reinstatement treatment in a different context from where they

Experiment 3A

Experiments 1 and 2 show that PND 23 rats exhibit reinstatement of an extinguished fear response and that this effect is dependent on the rats being tested in the same context where the post-extinction US was given. These experiments also show that PND 16 rats do not exhibit reinstatement of an extinguished fear response. This apparent developmental difference in post-extinction reinstatement is conceptually similar to our earlier findings that PND 16 rats fail to exhibit renewal of an

Experiment 3B

Experiment 3A shows that spontaneous forgetting in PND 16 rats is alleviated by the same reinstatement treatment that was ineffective at reversing extinction-induced performance deficits in PND 16 rats in Experiments 1 and 2, suggesting that the observed developmental difference in post-extinction reinstatement reflects a developmental dissociation in the extinction process. However, it should be noted that the mean level of freezing observed at test in rats in the No Reminder condition in

General discussion

The present study examined whether there are developmental differences in the reinstatement of an extinguished fear response following a post-extinction administration of the US. The results showed that a US Reminder treatment led to the recovery of extinguished fear in PND 23 rats, an effect that was context-dependent. In contrast, PND 16 rats did not exhibit reinstatement of extinguished fear following a US Reminder treatment. The failure to see post-extinction reinstatement in PND 16 rats

Acknowledgments

This research was supported by an Australian Postgraduate Award (JHK) and grant DP0346139 (RR) from the Australian Research Council. Reprint requests can be sent to Jee Hyun Kim, School of Psychology, University of New South Wales, Sydney Australia, 2052 ([email protected]).

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