We found that i.c.v. administration of NPY enhances the rate of within-session extinction and extinction retention for both contextual and cued aspects of conditioned fear and that antagonism of NPY Y1 receptors in the BLA results in decreased extinction retention, suggesting that endogenous NPY is important for the development of extinction.
Additionally, we have shown that administration of exogenous NPY inhibits the expression of fear-potentiated startle. The blockade of fear-potentiated startle was observed after i.c.v. and intra-BLA, but not intra-MeA, infusion of NPY. Moreover, we observed a dose-dependent reduction in baseline startle following i.c.v. administration of NPY. However, this was not seen in the amygdala, at least at the dose that was used, indicating the effects of NPY on fear-potentiated startle were not an artifact of a change in baseline startle.
Our finding that NPY infusion into the BLA, but not the MeA, inhibits the expression of fear-potentiated startle is consistent with previous findings. NPY is able to inhibit excitatory transmission in the amygdala (Molosh and Rainnie, 2007, unpublished observations). Electrophysiological data have shown that NPY agonists inhibit glutamate release in the hippocampus (Colmers et al., 1987
; Qian et al., 1997
). Pharmacological studies have shown that the expression of fear-potentiated startle is dependent on AMPA glutamate receptors in the BLA (Kim et al., 1993
; Walker and Davis, 1997
; Walker et al., 2005
). Therefore, it is likely that NPY is acting to decrease glutaminergic transmission in the BLA, thereby suppressing excitatory output from the amygdala and leading to the observed inhibition of fear-potentiated startle. We feel that the medial amygdala (MeA) is an ideal control region for these experiments, both because of its proximity to the BLA and because it expresses both NPY receptors and peptide (Chronwall et al., 1985
; Parker and Herzog, 1999
; Kopp et al., 2002
). Some evidence suggests that the central amygdala, a region responsible for output of information from the amygdala (Davis, 2000
), is also important for NPY signaling (Heilig et al., 1993
; Heilig, 2004
). Since the reduction in expression of fear-potentiated startle observed in the BLA was less than that seen with i.c.v. infusion, it is possible that another region helps mediate the i.c.v. effects. Future experiments can evaluate if NPY has effects in either the central amygdala or the bed nucleus of the stria terminalis, another output nucleus of the amygdala.
While we attempted to determine the receptor subtype mediating the reduction of fear-potentiated startle, our data remain inconclusive. We were not particularly surprised by the lack of effect of the Y1
receptor antagonist BIBO 3304 on the expression of fear-potentiated startle. Other studies have found no effect of BIBO 3304 alone on baseline behavioral measures, such as social interaction (Sajdyk et al., 1999
), suggesting the basal tone of NPY in the BLA may not be high enough to observe effects of an antagonist alone. This does not diminish the significance of our findings; rather, it suggests that while enhanced NPY signaling can inhibit the expression of conditioned fear, endogenous NPY is not necessary for normal expression of fear. Alternatively, it is possible that another NPY receptor subtype is involved. Y5
receptors in the BLA are implicated in anxiety-related behaviors (Sajdyk et al., 2002a
The finding that infusion of BIBO 3304 inhibits extinction of fear-potentiated startle suggests that the Y1
receptor modulates regulation of extinction learning in the BLA. This supports prior pharmacologic and genetic studies demonstrating that the Y1
receptor mediate anxiolytic effects of NPY (Kask et al., 2002
; Thorsell and Heilig, 2002
receptor agonists increase punished responding in a conflict test (Britton et al., 1997
) and antisense inhibition or a Y1
receptor antagonist blocks anxiolytic-like effects of NPY on the elevated plus-maze and social interaction (Heilig, 1995
; Sajdyk et al., 1999
). Bilateral administration of the nonpeptide Y1
receptor antagonist BIBP3226 into the amygdala results in increased anxiety-related behavior in the elevated plus-maze (Primeaux et al., 2005
). A recent study with NPY Y1
receptor null mice has shown that the Y1
receptor is necessary for anxiolytic-like effects of i.c.v. NPY on the elevated plus-maze (Karlsson et al., 2008
Because we typically see very little context conditioning using our parameters for measuring startle, we train and test animals in the same context. However, the results in Experiment 2 indicated considerable context conditioning and suggested that NPY was facilitating its extinction, thereby masking measurement of its possible effect on cue extinction. While the enhancement of extinction to contextual fear is an important finding, we wanted to determine whether or not central administration of NPY could also enhance extinction of cued fear. For this reason, we repeated the previous experiment but altered our testing context for extinction training and post-extinction testing (Experiment 7).
We no longer saw a difference in noise-alone startle amplitude between our NPY and vehicle groups in the Post-Ext test and were therefore able to compare percent fear in our groups since we no longer had differential shifts in our baseline startle values. shows that the NPY group had significantly lower percent fear-potentiated startle in the extinction retention test leading to the conclusion that exogenous administration of NPY can enhance both within-session and between-session extinction of fear-potentiated startle. Enhanced retention of extinction measured by lower levels of fear-potentiated startle could conceivably be explained by a lingering effect of NPY on fear-potentiated startle at test. However, we have observed in several other experiments that effects of NPY on baseline or fear-potentiated startle are gone by 48 hours after infusion. While these studies only test at 48 hours, we anticipate that these effects will persist long after this timepoint. Future experiments evaluating spontaneous recovery with this model can further elucidate the duration of the effect.
We chose to infuse an NPY antagonist, and not NPY peptide, into the BLA because antagonist studies provide a more direct approach for evaluating how endogenous systems function. Significantly higher levels of percent fear-potentiated startle during the extinction retention test were found following infusion of BIBO 3304 into the BLA. This demonstrates that blockade of NPY interferes with normal extinction and suggests that under normal conditions endogenous NPY Y1 receptors are activated during the extinction of conditioned fear. Remarkably, BIBO 3304 has no effect on baseline startle or expression of fear-potentiated startle when infused into the BLA (Experiment 4) thereby supporting the notion that our observed effects on extinction retention are not due to lingering effects of the drug itself but rather modulation of learning in the BLA.
Extinction of conditioned fear is dependent on NMDA glutamate receptors in the amygdala (Falls et al., 1992; Sotres-Bayon et al., 2007). GABAergic interneurons mediate inhibition of glutaminergic excitatory transmission in the basolateral amygdala (Rainnie et al., 1991
). Studies evaluating the electrophysiological effects of NPY in the arcuate nucleus indicate that NPY can inhibit GABAergic transmission (Acuna-Goycolea et al., 2005
). It is possible that NPY could be inhibiting a subpopulation of interneurons in the BLA, thereby disinhibiting the glutaminergic neurons responsible for extinction.
In conclusion, we believe that increases of NPY in the hippocampus when rats are exposed to a fearful context (Teppen, 2003
) or in the amygdala following a fearful cue (Gutman, Ressler, and Davis, unpublished observations) reflect its involvement in extinction and that its ability to improve resilience is based, in part, on its facilitation of extinction. This might also predict that enhancement of NPY Y1
receptor activation could be a potential adjunct for extinction-based psychotherapy, such as exposure therapy. Currently, the lack of available nonpeptide systemic NPY Y1
receptor agonists prohibits the direct clinical testing of this approach. An alternative strategy would take advantage of the presynaptic autoreceptor function of NPY Y2
receptors, which have been found to inhibit transmitter release (Michel et al., 1998
). Administration of antagonists to the NPY Y2
autoreceptor could lead to an endogenous enhancement of NPY Y1
receptor activation. This autoreceptor function may explain why administration of BIIE0246, a selective NPY Y2
receptor antagonist, has an anxiolytic-like effect in the elevated plus-maze in rats (Bacchi et al., 2006
). Genetic manipulation of the Y2
receptor further support this hypothesis, with Y2
receptor knockout mice exhibiting reduced anxiety in the open field and elevated plus-maze (Redrobe et al., 2003
; Tschenett et al., 2003
). Further studies are necessary to evaluate the merit of this approach in other models. Finally, it is conceivable that the ability of NPY to facilitate extinction observed in the present study might in some way be connected to the association between resilience and enhanced levels of NPY, such as those observed in soldiers under high stress and following trauma in individuals who do not develop PTSD (Morgan et al., 2000
; Yehuda et al., 2006