Extinction in preweanling rats does not involve NMDA receptors

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Abstract

Past research has shown that the N-methyl-d-aspartate receptor (NMDAr) is critically involved in the extinction of learned fear in adult rats with NMDAr antagonists impairing extinction retention and NMDAr agonists enhancing it. In the present study we examined the effects of the NMDAr antagonist MK-801 on extinction in the developing rat. In Experiment 1, rats were given pairings of a white-noise conditioned stimulus (CS) and a shock unconditioned stimulus (US) on postnatal day (P)16. An extinction session, where the CS was presented without the US, occurred on P17 or P24. Prior to extinction rats were injected with MK-801 or saline. All rats were tested for fear of the CS on P25, while drug free. Saline-treated rats exhibited good retention of extinction whether they were extinguished at P17 or P25. Rats treated with MK-801 exhibited impaired extinction retention but only if extinction occurred on P24. These findings show that extinction is NMDAr-dependent at P24 but NMDAr-independent at P17. Experiment 2 further examined the involvement of NMDA receptors in extinction at different stages of development by taking advantage of a finding from several recent studies on re-extinction. These studies have shown, in adult rats, a transition from NMDAr-dependent extinction to NMDAr-independent re-extinction. That is, if rats are trained, extinguished, re-trained to the same CS, and then extinguished again (i.e., re-extinction), then NMDA receptors are not required for extinction the second time. In Experiment 2, rats were trained to fear the CS at P16; this fear was extinguished at either P17 or P24. All rats were then re-trained to fear the CS at P25, re-extinguished at P26, and tested at P27. Prior to re-extinction, rats were injected with MK-801 or saline. Rats initially extinguished at P24, an age where NMDA receptors are involved in extinction, exhibited the transition to an NMDAr-independent re-extinction process. In contrast, rats initially extinguished at P17, an age where NMDA receptors are not involved in extinction, did not (i.e., these rats still exhibited an impairment in extinction retention if given MK-801 prior to re-extinction). Taken together, these findings demonstrate that a qualitatively different system mediates extinction early in life.

Introduction

Learned fear is extensively studied in the laboratory, most commonly through a Pavlovian conditioning procedure whereby an initially neutral conditioned stimulus (CS; e.g., a noise) is paired with an aversive unconditioned stimulus (US; e.g., a shock). As a result of the formation of an association between the two stimuli, subsequent presentations of the CS elicit various fear responses (e.g., freezing, potentiated startle). These learned fear reactions, however, can be reduced by repeatedly presenting the CS without the US, a procedure referred to as extinction. Fear extinction has attracted considerable interest over the past several decades, at both the behavioral and the neural levels of analysis (for review, see Myers and Davis, 2007, Quirk and Mueller, 2008). This interest is partly due to the theoretical importance of this phenomenon, and partly because understanding the processes by which fear is diminished is critical for the development of effective treatments for anxiety disorders (e.g., Pine, Helfinstein, Bar-Haim, Nelson, & Fox, 2009).

Early theoretical models 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 more widely accepted that the reduction in responding following extinction reflects learning of a CS-no US association that inhibits the expression of the original learned association (e.g., Bouton, 2002). The evidence for this view comes from numerous behavioral studies that show performance to an extinguished CS can recover without any re-training, but merely because of the passage of time (spontaneous recovery; e.g., Pavlov, 1927, Quirk, 2002), a change in environmental context (renewal; e.g., Bouton & King, 1983), or presentation of a pre-test stressor (reinstatement; e.g., Rescorla and Heth, 1975, Westbrook et al., 2002). Additional support for the claim that extinction involves new learning is provided by the finding that N-methyl-d-aspartate (NMDA) receptors are critically involved in extinction; NMDA has been shown to be involved in the intracellular molecular pathway that is critical for synaptic plasticity (Bliss and Collingridge, 1993, Kandel, 2001). NMDA receptor antagonists impair fear extinction (e.g., Baker and Azorlosa, 1996, Cox and Westbrook, 1994, Falls et al., 1992, Miserendino et al., 1990, Santini et al., 2001) while the NMDA receptor partial agonist d-cycloserine (DCS) facilitates fear extinction (e.g., Ledgerwood, Richardson, & Cranney, 2003: Walker, Ressler, Lu, & Davis; 2002).

However, recent studies suggest that extinction occurring early in development may not involve this new learning process but may rather be mediated by “unlearning” or “erasure”. For example, postnatal day (P)171 rats fail to exhibit either renewal or reinstatement of extinguished fear whereas P24 rats exhibit both renewal and reinstatement (Kim and Richardson, 2007a, Kim and Richardson, 2007b; also see Yap & Richardson, 2007). Further, P17 mice do not exhibit renewal or spontaneous recovery of an extinguished fear response whereas P24 mice do (Gogolla, Caroni, Luthi, & Herry, 2009). Most importantly for the present study, it has been reported that extinction is NMDA receptor-independent in P17 rats. That is, a pre-extinction injection of the NMDA receptor antagonist MK-801 had no effect on extinction retention in P17 rats whereas it impaired extinction retention in P24 rats (Langton, Kim, Nicholas, & Richardson, 2007). This failure of MK-801 to affect extinction in P17 rats was found across a range of doses (0.05, 0.10, and 0.20 mg/kg); further, injection of 0.1 mg/kg of MK-801 was found to affect acquisition of fear in both P17 and P24 rats (Langton et al., 2007). In other words, the lack of an effect of MK-801 on extinction in P17 rats cannot be attributed to rats this age being insensitive to this NMDA receptor antagonist.

Although the findings reported by Langton et al. (2007) are highly suggestive that extinction is independent of NMDA receptors early in life, it should be noted that the ages of conditioning and test, as well as extinction, differed in Langton et al. (2007). In other words, the developmental differences reported by Langton et al. could have been due the rats’ age at the time of fear conditioning or test, rather than their age at the time of extinction. For example, it might be the case that NMDA receptors are not involved in the extinction of fear acquired early in life (e.g., P16) but it is involved if the fear was acquired later in life (e.g., P23), regardless of the animal’s age at the time of extinction. Therefore, in the present study the rats’ age at the time of fear conditioning and test was kept constant, and the only age that varied was the age at the time of extinction. If extinction early in life is indeed independent of NMDA receptors, then similar results to those reported by Langton et al. should be found. However, if it is the age at conditioning or test that is critical, then results very different to those reported by Langton et al. (2007) will be obtained.

Section snippets

Subjects

All experiments used experimentally naive Sprague–Dawley derived rats, bred and housed in the School of Psychology at The University of New South Wales. Rats were 16 (±1) days of age at the start of both experiments. 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, and were treated according to the principles of animal use outlined in The

Experiment 1: role of NMDA receptors in extinction in early development

We recently reported that extinction in P17 rats is NMDA receptor (NMDAr)-independent while extinction in P24 rats is NMDAr-dependent (Langton et al. 2007). However, as noted earlier, groups also differed in terms of the age at the time of fear conditioning and test in that study. Specifically, rats extinguished at P17 were trained at P16 and tested at P18, whereas rats extinguished at P24 were trained at P23 and tested at P25. It may be the case that all rats trained at P16 exhibit

Experiment 2: role of NMDA receptors in re-extinction when initial extinction occurs at either P17 or P24

A number of recent studies have shown that extinction the second time (i.e., re-extinction) is different to extinction the first time. For example, while the amygdala is critically involved in extinction, it is not essential for re-extinction (e.g., Kim and Richardson, 2008, Laurent et al., 2008). Further, Langton and Richardson (2008) have shown that NMDA receptors are not involved in re-extinction. In that study adult rats were trained to fear a noise CS, and then had this fear extinguished

Discussion

Taken together, the experiments reported in this study show that extinction is NMDAr-independent early in life before becoming NMDAr-dependent later in life. In Experiment 1, rats extinguished at P17 exhibited extinction retention at test the following day regardless of whether they had been injected with saline or MK-801. In contrast, injecting MK-801 prior to extinction training impaired retention of extinction in P24 rats. This finding confirms the results reported in an earlier study by

Acknowledgments

This research was supported by Australian Research Council Discovery Project Grants (DP0666953 and DP0985554) to RR.

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