Re-exposure and environmental enrichment reveal NPY-Y1 as a possible target for post-traumatic stress disorder
Highlights
► Environmental enrichment (EE) and re-exposure (RE) enhanced behavioral recovery after foot shocks. ► RE normalized the foot shock (FS)-induced changes in glutamate receptor mRNAs in the amygdala. ► EE and RE normalized the FS-induced increase in CRF-R1 mRNA in the amygdala. ► RE and EE increased NPY-Y1 receptor mRNA in amygdala. ► Infusion of a NPY-Y1 agonist in the amygdala reduced FS-induced anxiety.
Introduction
Post-traumatic stress disorder (PTSD) patients show symptoms, such as intrusive memories, avoidance, and an exaggerated response to cues that have just a slight resemblance to the trauma (Hackmann et al., 2004; Reynolds and Brewin, 1998). These symptoms are considered consequences of associative learning, such as classical conditioning, operant conditioning, (re)consolidation, and generalization (Grillon et al., 1996; Solomon et al., 1987). In addition, PTSD also leads to symptoms that are caused by the trauma but are not the result of associative fear learning. These symptoms are rather the result of fear incubation (Boyd, 1981; Eysenck, 1968; Pickens et al., 2009b) (resulting in enhanced reaction to situational reminders over time without further conditioning) or sensitization (Post et al., 1995; Stam, 2007; Stam et al., 2000) (resulting in an enhanced sensitivity towards (neutral) stressful stimuli that have no association with the trauma). These forms of non-associative learning can lead to persistent symptoms of anxiety and hyperarousal that may include hypervigilance, exaggerated startle response, and emotional numbing (Pamplona et al., 2010; Siegmund and Wotjak, 2007a, b; Stam et al., 2000). On top of these symptoms there is a high comorbidity with depression (77%) and generalized anxiety (34%) (Kean et al., 2007). Exposure-based behavioral therapy is used to acquire extinction for trauma-associated cues, but it also relieves the non-associated symptoms (Moulds and Nixon, 2006; Paunovic and Ost, 2001; Powers et al., 2010; Yehuda, 2002). The extinction process has been extensively studied both in animals and in humans (Bouton and King, 1983; Myers and Davis, 2007; Quirk et al., 2010). These studies have shown, for instance, that glutamatergic N-methyl d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the amygdala play an important role in fear extinction (Davis, 2006; Sotres-Bayon et al., 2007; Yamada et al., 2009).
The mechanisms behind the reduction in stress sensitivity following re-exposure therapy are less clear. Since exposure therapy reduces both types of symptoms in PTSD patients, we hypothesized that glutamate receptors may also play a role in the reduction of stress sensitivity. In addition, the neuropeptide Y (NPY) and corticotropin-releasing factor (CRF) systems may be involved. NPY is an endogenous anxiolytic peptide that exerts its effect in the amygdala via NPY-Y1 receptors (Y1) (Heilig et al., 2004; Kask et al., 2002; Sajdyk et al., 1999). Various studies suggest a role for NPY in PTSD. For example, NPY is involved in incubated and non-incubated fear in the rat (Pickens et al., 2009a). In PTSD patients cerebrospinal fluid levels of NPY are decreased (Sah et al., 2009). CRF is involved in stress-related behavior and has anxiogenic properties upon central administration (Bijlsma et al., 2011; Britton et al., 1982; Swerdlow et al., 1989). As suggested by Heilig, the balance between CRF and NPY in the amygdala modulates the state of anxiety/fear of an animal (Heilig, 2004). CRF-R1 is involved in the consolidation of fear memories after foot shock (Thoeringer et al., 2012), while blockade of this receptor inhibits the consolidation of stress effects in the predator stress model of PTSD (Adamec et al., 2010).
In this study we used the inescapable foot shock (IFS) model for PTSD (Hendriksen et al., 2010) to investigate putative mechanisms underlying the behavioral recovery following re-exposure therapy. IFS instates a long-lasting fear towards the traumatic context, but also an aggravated response to (neutral) anxiogenic situations that are not directly associated with the induction of the traumatic event (Geerse et al., 2006a; Hendriksen et al., 2010; Stam et al., 2000; van Dijken et al., 1992a, van Dijken et al., 1992b), accompanied by long-lasting changes in the hypothalamic-pituitary-adrenal (HPA) axis (van Dijken et al., 1993). We used sudden cessation of a background noise (stress of sudden silence) as a neutral (unconditioned) stressor to measure enhanced stress sensitization (Buwalda et al., 2001; Hendriksen et al., 2010; Rogalska et al., 2006; van Dijken et al., 1992a, van Dijken et al., 1992b). Recently we showed that environmental enrichment (EE) stimulates recovery of enhanced stress sensitization after IFS (Hendriksen et al., 2010).
Here we report that repeated re-exposure to the context in which the animals received the foot shocks attenuated their fear for the shock context, but also their non-trauma-associated anxiety. Effects of RE on stress sensitivity were compared with those of EE. Next we tried to connect the reduced anxiety following RE with changes in expression of selected genes in the amygdala. Pronounced changes in gene expression were found for the AMPA receptor subunits GLuK3, GluK4 and CRF-R1 and NPY-Y1. Next we investigated the behavioral consequences of some of these molecular changes by intra-amygdala administration of an AMPA/kainate receptor antagonist or an NPY-Y1 agonist.
Section snippets
Ethics statement
All animals were handled in strict accordance with good animal practice as defined by the Ethical Committee for Animal Research of Utrecht University, and all animal work was approved by the Ethical Committee for Animal Research of Utrecht University (DEC-ABC) DEC nr: 2007.I.01.010/vv-1-3 and 2009.I.12.108.
Animals
Male Sprague Dawley rats (Harlan, The Netherlands) were 8–9 weeks old and weighed 220–250 g on arrival. We started the experiments after one week of acclimatization of the animals to the
Re-exposure and context-associated anxiety
Fig. 3A shows the effects of extinction training on the latency time to enter the dark compartment. One week after the foot shocks, the latency times of the IFS animals (N = 12) to enter the shock compartment was 134.5 ± 9.0 s, while controls (N = 12) entered within 4 s (day 0: H(1) = 16.936, p < 0.01). After two days of re-exposure (RE) the latency time of the IFS animals was back to control values (H(1) = 1.843, p > 0.05) and stayed at this level for the rest of the RE procedure (day 8:
Discussion
In this study we explored the mechanisms underlying the therapeutic effect of re-exposure-based behavioral therapy in the IFS animal model for PTSD. We especially focused on the more generalized anxiety of the IFS animals, which is thought to be the result of increased stress sensitivity. Imaginal or prolonged exposure is in most cases the core component of the PTSD treatment (Kaplan et al., 2011; Noordik et al., 2010). It refers to reliving the traumatic memories (in vitro flooding) or to the
Conclusion
Here we described two different non-pharmacological interventions in the IFS model, resembling behavioral therapy for humans, that reveal NPY-Y1 as a putative target for PTSD. Although we did not prove that NPY-Y1 is obligatory for the anxiolytic effect of the behavioral treatments, we did validate its anxiolytic efficacy in IFS animals. The mRNA expression data of the CRF-R1suggest that this receptor might also be involved in the reduction of IFS-induced anxiety following RE or EE. We did not
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