Discussion

A number of studies have investigated the effects of early life aversive experience on adult behavior. Most, however, have focused on maternal deprivation or other

stressors(Callaghan & Richardson, 2011; Guijarro et al., 2007; Kosten, Kim, & Lee,

2012; Madruga, Xavier, Achaval, Sanvitto, & Lucion, 2006; Stevenson, Spicer, Mason,

& Marsden, 2009). The current work, similarly to Jones and Monfils (2016), directly examined the retention of early fear conditioned memories, and their enduring effects on fear re-acquisition in adulthood. Our results show that early predictable or unpredictable aversive learning at P17 or P25 differentially modulate fear acquisition and retention in adulthood.

First, paired conditioning at P17 or P25 lead to increased freezing 24-hours later.

Yet, in-line with Jones and Monfils (2016), as well as Travaglia et al. (2016), fear memory acquired at P25, but not P17, endured into adulthood, as shown by increased levels of freezing to the context (un-paired early life conditioning) and the cue (paired early life conditioning). Interestingly, experiment one also indicates predictability (paired vs. unpaired) of fear exposure at P17 and P25 yielded differential consequences on fear responding after varying adult conditioning procedures. Both paired and unpaired conditioning at P17 potentiated paired conditioning in adulthood, during extinction and the subsequent long-term memory test. While paired conditioning at P25 potentiated both paired and unpaired conditioning in adulthood, unpaired training at P25 off-set adult paired conditioning.

While results from experiment two replicate those of others in the field, indicating an initial and immediate fear memory when tested within 24-hours after early life

conditioning (Kim et al., 2009; Rudy, 1993; Travaglia, Bisaz, Sweet, et al., 2016). The variation in the length from conditioning to test in experiment one may contribute to differential responding seen after a longer delay (months), in which rats conditioned at

P17 do not show evidence of explicit memory in adulthood (i.e., they do not freeze). An effect Spear (1979) describes is due to the fact that memories acquired during infancy are easier to “forget”. For example, Akers and colleagues (2012) show that infant contextual fear memory is no longer expressed as soon as 1-week after training, whereas when trained at P30 or P60 freezing was still elicited to the context at least 1-month later. In line with our behavioral timeline, Coulter and colleagues (1976) found that infant P11-P16 animals show complete forgetting 42-days after cued fear conditioning, an effect not seen in the older groups, for which neurological immaturity may underlie the forgetting of earlier events.

Though we did not examine the neural mechanisms of the differential effects of conditioning at P17 vs. P25, there are clues from the literature. One possibility to explain the phenomenon of infantile amnesia or forgetting that we observed in the P17 rats, is the rapid birth of hippocampal cells during early life, which may suggest a lack of maturity in this critical brain region (Frankland, Köhler, & Josselyn, 2013; Josselyn &

Frankland, 2012). Importantly, the period between P17 and P24 appears to undergo critical changes in other memory systems as well. Previous work has shown that markers of neuronal activity, plasticity, synaptic maturation, neurite connectivity and myelination, as well as AMPA receptors themselves are differentially expressed after conditioning in P17 versus P24 rats, but that P24 and adult rats share similar responses in AMPA receptors and plasticity markers (Travaglia, Bisaz, Cruz, & Alberini, 2016; Travaglia, Bisaz, Sweet, et al., 2016). These studies emphasize that differing behavioral responses may arise from recruitment of different neural systems during acquisition.

The present study also found contingency of early life conditioning (paired or unpaired) differentially modulated outcomes after reconditioning in adulthood. An effect shown by Sevelinges and colleagues after early life (P8 to P12) fear experience

(Sevelinges et al., 2007, 2008). Our results support the importance of contingency as P25 rats exposed to early paired conditioning show a potentiation in freezing after adult paired conditioning, while unpaired experience at the same age blocks this response resulting in no difference initially to previously naive controls but at block three they show significantly less freezing. These data suggest that early memory can modulate adult conditioning, but those effects are dependent on the contingency of early life events. This may be particularly important when we consider the fact that the un-pairing procedure has been shown to produce safety learning to a cue (Ostroff, Cain, Bedont, Monfils, & Ledoux, 2010; Rescorla, 1969; Rogan, Leon, Perez, & Kandel, 2005). In particular, learned irrelevance, a phenomenon in which unpaired training subsequently retards paired conditioning, is not present at P17 or P20, but emerges by P25 (Rush, Robinette, & Stanton, 2001; Stanton, Fox, & Carter, 1998). This reduction in responding after early unpaired then paired training was seen in our P25 rats, however contrary to previous findings, unpaired training at P17 followed by paired training in adulthood potentiated fear responses. The potentiation of fear after P17 conditioning however may be considered a generalized response, as paired conditioning in early life and adulthood also produced an increase in fear responding. This effect is counter to Sevelinges’s studies (2007, 2008) in which paired and not unpaired groups at P8 -12 lead to an attenuation rather than potentiation of freezing responses upon reconditioning. This

disparity may arise however, from the earlier developmental time point used during initial conditioning and the paradigm used. Specifically, the odor-shock paradigm used in Sevelinges et al. (2007, 2008) has also been shown to produce an odor preference at earlier developmental time points, P8-9 (Sullivan, Landers, Yeaman, & Wilson, 2000), which may then offset the new aversive learning. In either case these and other previous findings support the hypothesis that long-lasting memory traces acquired early in life can be recruited in adulthood (Stella Li, Callaghan, & Richardson, 2014; Schäble et al., 2007).

One avenue for future research should continue the exploration of ways to reduce the potentiation of fear. The retrieval extinction paradigm shows promise in this regard (Jones & Monfils, 2016). Li and Richardson (2013) found that 14-days after P17 conditioning rats elicit no memory for the conditioned cue (similar to current findings), but that the application of MK-801 (a N-methyl-D-aspartate receptor [NMDAr] inhibitor) blocked the reacquisition of fear. This approach however may not transfer to P25

conditions, as the lack of memory by P31 may be the key, since additional work has shown acquisition (Fanselow & Kim, 1994; Laurent & Westbrook, 2009; Miserendino, Sananes, Melia, & Davis, 1990), but not re-acquisition/reconditioning have been found to be NMDA independent (Roesler et al., 1998; Sanders & Fanselow, 2003; Tayler et al., 2011). The fact that P17 rats do not show a memory for the cue in adulthood makes a cue-targeted approach challenging for translation into clinical populations. Thus, future work should explore alternative and potentially indirect approaches that may work independent of timing of early life aversive or traumatic experience.

In the present work, emotional memory in early life was found to persist and impact adult learning behavior. We show that early predictable or unpredictable aversive experience at P17 or P25 differentially modulates fear memory and reconditioning in adulthood. The most challenging of these findings, with respect to translation to the clinic, is that rats conditioned with either paired or unpaired procedures at P17 exhibiting no initial fear memory but a potentiated response after adult paired procedures,

suggesting a more generalized response to aversive experiences in early life. These findings underscore the necessity to develop both direct and indirect approaches to reduce the persistence of fear irrespective of when in early life fear exposure occurred.