We hypothesized that adolescent rats would show poorer extinction retention (ie, greater fear relapse) when tested 24
h after extinction training compared with both younger and adult rats. This hypothesis was based on evidence that PFC (1) is critical for extinction retention (Herry and Garcia, 2003
; Quirk et al, 2000
; Milad and Quirk, 2002
), and (2) undergoes substantial restructuring during adolescence (Blakemore and Choudhury, 1999
), to the point that it might even be seen as a ‘natural lesion'. Experiment 1 confirmed this hypothesis, with adolescent rats exhibiting significantly higher levels of freezing at test compared with both younger (ie, preadolescent) and older (ie, young adult) rats (also see Kim et al, in press
). Experiment 2 then showed that this impairment in extinction retention in adolescent rats could be alleviated by injecting 15
mg/kg of DCS immediately after the extinction session; a lower dose of DCS (5
mg/kg) was ineffective. Experiment 3 replicated the finding that DCS enhances extinction retention in the adolescent rat, and also showed that it had to be given shortly after the extinction session to be effective; ie, injecting DCS 4
h after extinction had no effect. Finally, experiment 4 replicated the DCS finding once again, and also showed that doubling the amount of extinction training led to a similar improvement in extinction retention in the adolescent rat.
The results of experiments 1–4 consistently show that adolescent rats fail to retain an extinction memory over a 24-h period. Across the four experiments, adolescent rats exhibited a comparable and essentially complete recovery of fear at test the following day (ie, 105, 90, 100, and 106% recovery of fear, as compared with the first block of extinction trials, in experiments 1–4, respectively). It is important to note that the present results also show two ways in which the impaired extinction retention in adolescent rats can be alleviated. First, systemic injection of the NMDA receptor partial agonist DCS immediately after the extinction session led to enhanced retention (ie, lower levels of freezing; experiments 2–4). This result could be because of one of several mechanisms. One possible mechanism for the observed effect of DCS on retention of extinction in adolescent rats is that it enhanced the activity of NMDA receptors in the PFC. In a recent study, we examined phosphorylated MAPK (pMAPK) in the PFC of 24, 35, and 70 day olds, using the same procedures as in this study. We measured pMAPK in that study because previous research had shown that extinction involves activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway in the mPFC (Kim et al, 2009
), and that postextinction blockade of MAPK/ERK in the mPFC disrupts long-term extinction (Hugues et al, 2004
). In our recent study, levels of pMAPK in the PFC 1
h after extinction training was elevated in 24- and 70-day-old rats, but not in 35-day-old rats (Kim et al, in press
). This finding suggests that PFC is not activated in the adolescent rat when the current training/extinction procedures are used. This could be the reason why the adolescent rat exhibits such poor extinction retention. Injecting DCS immediately after extinction may lead to an increased level of pMAPK in the PFC of adolescent rats, which then leads to enhanced retention of extinction. Some support for this notion is provided by a recent study, in which infusion of the NMDA antagonist CPP directly in the PFC impaired fear extinction retention in adult rats (Burgos-Robles et al, 2007
). In addition, a recent fMRI study found that administration of DCS enhanced PFC activity during symptom provocation in spider phobics (Aupperle et al, 2009
). Another possible mechanism for the observed enhancing effects of DCS on extinction retention in adolescent rats is that the DCS enhances activity of NMDA receptors in the amygdala, leading to a stronger extinction memory. It is well established that the amygdala has a high density of NMDA receptors (Monaghan and Cotman, 1985
), and numerous studies have shown the importance of these particular receptors in extinction retention by directly infusing either NMDA receptor antagonists (Falls et al, 1992
) or the partial receptor agonist DCS (Ledgerwood et al, 2003
; Walker et al, 2002
) into the amygdala.
A second way in which the impaired extinction retention in adolescent rats can be alleviated is by doubling the number of extinction trials from 30 to 60. Adolescent rats treated in this way showed comparable low levels of freezing (ie, good retention of extinction), as did adolescent rats given DCS immediately after 30 extinction trials (experiment 4). This finding is similar to the finding that adult rats with lesions of the PFC can eventually exhibit retention of extinction if given extra extinction training (Lebron et al, 2004
), and the finding that doubling the number of extinction trials in adult rats leads to a comparable improvement in extinction retention as does injecting DCS (Ledgerwood et al, 2005
). It will be interesting to determine whether the mechanism mediating this improvement in extinction retention is the same, or different, to that mediating the improvement produced by DCS.
The findings from the present study showing that adolescent rats show markedly poorer retention of extinction, compared with both younger and older rats, may mean that treatment gains made in adolescents are much more vulnerable to relapse. Importantly, the results reported here also suggest that the poor retention of extinction in adolescent rats can be completely overcome by either doubling the amount of extinction training or by injecting the NMDA receptor partial agonist DCS immediately after extinction. Although some clinical research has failed to find a benefit of administering DCS (Storch et al, 2007
), several other studies have found that DCS enhances treatment outcomes in adults suffering from various anxiety disorders (Guastella et al, 2008
; Hofmann et al, 2006
; Ressler et al, 2004
; Wilhelm et al, 2008
; see Norberg et al, 2008
, for meta-analysis). The results of this study, if they can be extended to humans, suggest that DCS might be an effective pharmacological adjunct to exposure-based therapy in adolescent populations as well.
Future Research Directions
In all four experiments in this study, adolescent rats (ie, 35 days of age) exhibited poor retention of extinction over a 24-h interval. That is, despite showing low levels of fear at the end of the extinction training session (see panel (c) in all four figures), these rats exhibited substantially higher levels of freezing at test the next day (also see Kim et al, in press
). This effect was not observed in either younger (ie, 24 days of age) or older (ie, 70 days of age) rats (experiment 1; also Kim et al, in press
). We did not test to determine the exact age that this impaired extinction retention first emerges or when it no longer occurs, but other research has shown that activity in the infralimbic PFC is critically involved in extinction retention in rats aged 24–30 days (Santini et al, 2008
). On the basis of that finding, one would predict that impaired extinction retention first emerges sometime after 30 days of age. However, determination of the exact time when this impairment first occurs will have to await future studies, as will the determination of the exact age at which the impairment is no longer observed.
In addition, future experiments are needed to determine the location in the brain where DCS is having its effects. As noted earlier, the enhancement of extinction retention in adolescent rats given DCS could be because of its action in either the PFC or the amygdala (or both). In the present study, DCS was given systemically, so conclusions about the neural bases of the observed effects are not possible. On the basis of our hypothesis that the impaired extinction retention observed in adolescent rats is due to the restructuring of the PFC during this period of development, we predict that infusions of DCS into the PFC will enhance extinction retention in adolescent rats. In the present study, we also found that doubling the number of extinction trials enhanced extinction retention in adolescent rats. Future experiments will be needed to determine whether this manipulation affects extinction retention through the same mechanism(s) as does DCS. For example, do both treatments affect activity in the PFC and therefore lead to enhanced extinction retention? In that regard, we have found that doubling the number of extinction trials does lead to increased number of pMAPK immunoreactive cells in the infralimbic cortex of adolescent rats (Kim et al, in press
). We do not currently know whether administering DCS immediately after extinction will have a similar effect.
It will also be important to examine whether DCS, or doubling the number of extinction trials, enhances extinction retention in female rats, as all the animals used in these experiments were male. This issue is particularly important, given that it is well documented that the prevalence of anxiety disorders in humans is often as much as twice as high for females compared with males (Kessler et al, 2005
), particularly in adolescence (Lewinsohn et al, 1998
). Finally, it will be important to determine whether similar impairments in extinction retention are also observed in human adolescents. If they do, then such findings would have substantial implications for our understanding of treating anxiety disorders during this period of development. However, irrespective of the results of those future studies, the findings reported here clearly document that there is a marked impairment in extinction retention in adolescent rats.