Phosphorylation of mitogen-activated protein kinase in the medial prefrontal cortex and the amygdala following memory retrieval or forgetting in developing rats

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Abstract

We examined neuronal correlates of forgetting in rats by detection of phosphorylated mitogen-activated protein kinase (pMAPK) in the medial prefrontal cortex (mPFC) and amygdala. In Experiment 1, postnatal day (P)23 and P16 rats received paired noise CS-shock US presentations. When tested immediately after conditioning, P23 and P16 rats exhibited similar levels of conditioned fear; when tested after 2 days, however, P16 rats showed poor CS-elicited freezing relative to P23 rats. In Experiment 2, P16 and P23 rats received either paired or unpaired CS–US presentations, and then were tested 48 h later. Consistent with Experiment 1, P16 rats showed forgetting whereas P23 rats exhibited good retention at test. Additionally, unpaired groups showed poor CS-elicited freezing at test. Immunohistochemistry showed that P23 and P16 rats given paired presentations exhibited significant elevation of pMAPK-immunoreactive(ir) neurons in the amygdala compared to rats given unpaired presentations. That is, MAPK phosphorylation in the amygdala tracked learning history rather than behavioral performance at test. In contrast, only the P23-paired group showed an elevated number of pMAPK-ir neurons in mPFC, indicating that MAPK phosphorylation in the mPFC tracks memory expression. Different test-perfusion intervals were employed in Experiment 3, which showed that the developmental dissociation in the pMAPK-ir neurons observed in the mPFC in Experiment 2 was not due to age differences in the rate of phosphorylation of MAPK. These findings provide initial evidence suggesting that while the mPFC is involved in memory retrieval, MAPK phosphorylation in the amygdala may be a persisting neural signature of fear memory.

Highlights

► We examine MAPK activation in the brain of rats that remember or forget fear. ► Remembering was associated with significant phosphorylation of MAPK in the mPFC. ► There was MAPK activation in the amygdala even after forgetting.

Introduction

Over the past decade, considerable progress has been made in delineating the neural bases of learning and memory (Kandel, 2001). In contrast, less attention has been paid to the neural bases of forgetting, despite its clinical significance. For example, an inability to forget can lead to anxiety disorders such as post-traumatic stress disorder, whereas excess forgetting is observed in dementia and Alzheimer’s disease. One reason for the relative neglect of the neurobiological study of forgetting is that spontaneous forgetting is difficult to observe in animals; in one study adult rats were shown to remember a learned fear response for 12 months (Gale et al., 2004). However, spontaneous forgetting is easily observed in infant animals (Campbell & Spear, 1972). Young rats, for example, exhibit much more rapid forgetting than do older rats, a phenomenon referred to as infantile amnesia. Typically, no age differences in retention are observed when testing occurs soon after training, indicating that animals of all ages are equally able to acquire and express memory. However, when testing occurs after a delay, retention increases dramatically with age. This general finding has since been replicated numerous times, and is of substantial biological significance because it is an ubiquitous phenomenon that occurs in all altricial species, including humans (see Campbell and Spear (1972), for review).

Interestingly, infantile amnesia reflects a failure to retrieve the fear memory at test because various pretest “reminder” treatments are able to reinstate the memory. For example, a single pre-test shock unconditioned stimulus (US) can reverse infantile forgetting (Kim and Richardson, 2007a, Kim and Richardson, 2007c, Spear and Parsons, 1976). Infantile amnesia is also attenuated via modulations of γ-aminobutyic acid (GABA) neurotransmission. Specifically, a pre-test injection of the GABAA receptor partial inverse agonist FG7142 attenuated forgetting in infant rats (Kim et al., 2006, Kim and Richardson, 2007b). From this, it is clear that young animals are capable of encoding and storing a memory, but are impaired in retrieving that memory. However, the neural mechanisms underlying infantile amnesia are largely unexplored. Therefore, the present study investigated the neural correlates of memory retrieval and forgetting in developing rats. We first gave white-noise conditioned stimulus (CS) and shock US pairings to postnatal day (P)16 and P23 rats and tested them for memory retention. Following test, phosphorylation of mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK) in the amygdala and the medial prefrontal cortex (mPFC) was analyzed by immunohistochemistry. MAPK is a part of the intracellular signaling pathway involved in activity-dependent modulation of synaptic strength in various brain areas including the amygdala and the mPFC (English and Sweatt, 1997, Herry et al., 2006, Otani et al., 1999). The basolateral amygdala (BLA), in particular, was examined in the present study because it is a well-known critical structure for learned fear (Davis, 2000, LeDoux, 2000). The medial amygdala (MeA) was also examined because of its importance for unlearned fear and/or general anxiety (Davis & Whalen, 2001). As previous research suggests that infantile amnesia is a retrieval failure, there may be evidence of long-term cellular activity in the MeA as residual anxiety despite the absence of behavioral expression of memory. The mPFC was examined because of recent evidence suggesting that expression of learned fear is mediated by the mPFC (Corcoran and Quirk, 2007, Sotres-Bayon and Quirk, 2010).

Section snippets

Subjects

Experimentally-naive Sprague–Dawley derived rats, bred and housed in the School of Psychology, The University of New South Wales, were used. Rats were either 16 (±1) or 23 (±1) days of age at the start of 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 covered by a wire lid. Animals were maintained on a 12 h light/dark cycle (lights on at 6 am) with food and water available ad libitum.

Experiment 1

Infantile amnesia refers to the more rapid spontaneous forgetting observed in young compared to adult animals. Campbell and Campbell (1962) first demonstrated that P18 rats showed complete forgetting after 7 days whereas P100 rats showed excellent retention even after 42 days. When these rats were tested immediately after training, no differences in memory were found. Recently, Kim and Richardson, 2007a, Kim and Richardson, 2007b, Kim and Richardson, 2007c reported that P16 rats trained with 2

Experiment 2

Experiment 1 demonstrated that over a 48-h retention interval, P16 rats showed substantial forgetting of conditioned fear whereas P23 rats did not. In Experiment 2, P16 and P23 rats received either paired or unpaired presentations of the CS and the US; all rats were then tested 48 h later. Fifteen-minutes following test, we examined MAPK/ERK phosphorylation in the prelimbic cortex (PrL), infralimibic cortex (IL), BLA, and MeA. Phosphorylation of MAPK is a part of the intracellular signaling

Experiment 3

In Experiment 2, it was observed that when tested 48 h after training, P23 rats show good retention whereas P16 display forgetting. This retrieval of memory in the P23 rats was associated with an increase in the number pMAPK-ir neurons in the mPFC. However, it may be the case that the observed developmental dissociation in the phosphorylation of MAPK in the mPFC is due to age differences in the timing of MAPK phosphorylation following test. Experiment 3 examined this possibility by perfusing the

General discussion

In this study, we investigated the neural correlates of infantile amnesia in rats. Experiment 1 showed that P16 and P23 rats exhibited comparable levels of CS-elicited freezing when tested immediately after training. Forty-eight hours later, however, P23 rats exhibited good retention whereas P16 rats showed substantial forgetting (i.e., infantile amnesia was observed). In Experiment 2, P16 and P23 rats received either paired or unpaired CS–US presentations, and then were tested 48 h later.

Acknowledgments

This research was supported by an Australian Postgraduate Award (JHK), Howard Florey Medical Research Foundation Fund (JHK), and Discovery grants from the Australian Research Council to RR (DP0666953) and GM and RR (DP0880854). We thank Melissa Onden-Lim for assisting in collecting the data for Experiment 1.

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    Present address: School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW 2678, Australia.

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